Selecting substances for treating glucocorticoid-mediated inflammation or immune diseases using Tripterygium wilfordii Hook F extracts

ABSTRACT

The present invention provides for the use of Tripterygium wilfordii Hook F extracts and purified components thereof in the treatment of inflammation or an immune disorder with concomitant lack of steroidal effect. Extracts of this plant (T2) bound to the glucocorticoid receptor and competitively inhibited glucocorticoid mediated cellular processes, such as dexamethasone binding to the glucocorticoid receptor, glucocorticoid mediated activation of target genes, dexamethasone dependent cellular growth, with concomitant inhibition of cyclooxygenase-2 induction and inflammatory processes such as the production of prostaglandin E 2 . The T2 extract components triptolide and tripdiolide were effective inhibitors. The particular advantage provided by the methods herein is the treatment or prevention of inflammation and the concomitant lack of steroidal agonist effects and NSAID side effects. Conditions treatable by the present methods include inflammation and immune disorders including autoimmune disease.

The government has rights in the invention developed in parentapplications, U.S. Ser. Nos. 07/862,836 (filed Apr. 3, 1992) and07/494,113 (filed Mar. 14, 1990), as research relevant to thedevelopment thereof was supported by a grant from the United Statesgovernment, NIH grant AR-36169.

This application is a divisional of U.S. patent application Ser. No.08/455,906 filed on May 31, 1995, now U.S. Pat. No. 5,616,458, which isa continuation-in-part of U.S. patent application Ser. No. 08/168,980filed on Dec. 17, 1993, now U.S. Pat. No. 5,580,562, which is acontinuation-in-part of U.S. patent application Ser. No. 07/862,836filed on Apr. 3, 1992, now U.S. Pat. No. 5,294,443, which is acontinuation-in-part of U.S. patent application Ser. No. 07/494,113filed on Mar. 14, 1990, now abandoned.

BACKGROUND OF THE INVENTION

Rheumatoid arthritis (RA) is a chronic inflammatory disease of uncertainetiology. Since the cause is unknown, treatment has been directed atsuppressing the signs and symptoms of chronic inflammation. Althoughmany agents have been documented to decrease pain and swellingtemporarily, none has been shown to have a major impact on the course ofthe disease. While therapeutic modalities have been developed fortreatment of this disease¹⁻⁴, uniform and persistent suppression of thiscondition has not been reported. Although current approaches remainpromising, alternative means of drug development seem warranted andcould yield not only new and effective treatment modalities, but alsoprovide new insights into disease pathogenesis that could serve as thebasis of future therapeutic innovations.

An area to search for new therapeutic interventions for different formsof arthritis, and particularly RA and other autoimmune diseases, is thatof traditional Chinese medicines.

One of these traditional medicines is from Tripterygium wilfordii HookF, a shrub-like vine from the Celastraceae family⁵. Tripterygiumwilfordii Hook F is known to contain a number of constituents, some ofwhich appear to be toxic⁶. It is known that the leaves, stem, flowers,and the skin of the roots are poisonous and that ingestion can causedeath⁷⁻⁹. In contrast, the woody portion of the roots of the plant ismuch less toxic. An extract of Tripterygium wilfordii Hook F preparedfrom the root of the plant, designated T₂, has been described in theChinese literature for the treatment of autoimmune diseases¹⁰⁻²⁶. Thepreparation appeared to contain therapeutic components, and to have areduced toxicity compared to other available preparations of the plant.

The T₂ extract has been evaluated in a double-blind placebo controlledcross-over study involving 70 RA patients, these patients having had amean duration of RA of 6 years¹⁰⁻¹¹. Significant improvement in avariety of clinical parameters, particularly ESR, CRP, and Rheumatoidfactor titers, was noted after 12 weeks of therapy in the experimentalgroup compared with either baseline measurements or the placebo treatedgroup. Of the patients treated, 82-93% noted improvement in differentclinical criteria or laboratory correlates of inflammation. Animmunosuppressive activity of T₂ may be inferred from the finding thattreatment induced inhibition of the production of IgM and IgM rheumatoidfactor by the patients' peripheral-blood mononuclear cells in vitro⁷.Toxicity, which consisted primarily of skin rash, gastrointestinalcomplaints and amenorrhea, was reportedly of a generally mild nature,and reversible with cessation of therapy.

The Chinese experience suggested that a daily dosage of 0.8-1.5 mg/kg ofT₂ was relatively safe and effective. Acute and chronic toxicity studieshave been carried out with T₂ in China using a variety of animal models.The LD₅₀ of T₂ in mice was found to be 159.7±14.3 mg/kg²⁷. The majorchronic toxicity noted in rats administered 30 mg/kg for 90 days wasazoospermia and decrease in testicular weight²⁷. Lower dosages of T₂ didnot cause decreases in testicular weight. The toxicity studies,therefore, suggested that T₂ exhibited a reasonable safety index andshould be able to be administered to patients safely.

Research has begun in China to determine the spectrum of activity ofvarious preparations of T. wilfordii. According to the reported resultsof these studies, extracts of TwHF were able to inhibit E-rosetteformation by guinea pig T cells, mitogen induced IL-2 production bymouse T cells and antigen stimulated migration of rat lymphocytes²⁸, 29.Components of T. wilfordii hook F known as triptonide and triptolidehave been reported to inhibit the proliferation of lymph cells inducedby concanavalin A³⁰. Chloroform/ethanol extracts of the plant, referredto as T₂ in the literature, have been described as having significantactivity in vivo against certain mouse leukemias and in vitro againstcells derived from human carcinomas³¹. The capacity of T₂ to suppress anumber of animal models of autoimmune disease, including adjuvantarthritis and experimental allergic encephalomyelitis, has beenreported²⁸⁻²⁹,³²⁻³⁶. Large concentrations of T₂ preparations (30 mg/kg)have been reported to suppress delayed-type hypersensitivity reactivityin mice and may also suppress graft-versus-host disease, as well as skinand heart allograft rejection⁶⁻³². It remains unclear whether lower,more pharmacologically appropriate concentrations would also exerttherapeutic effects in these animals, however.

The T₂ examined in the Chinese literature is a crude extract containinga mixture of materials, including various glycosides, alkaloids, andditerpenoids. The active principle, however, has not yet beenidentified. A few components have been purified, including triptolide,wilfordine, and related compounds, but no particular purified componentwhich accounts for the therapeutic or immunosuppressive activity of T₂exists⁴⁰. High concentrations of triptolide were reported to suppress Band T lymphocyte proliferation and interleukin-2 production by mousespleen cells⁴¹. However, the concentrations of the T₂ used weresufficiently high that significant nonspecific toxicity undoubtedlyoccurred.

A number of pharmacologic agents have been used to treat rheumatoidarthritis and other inflammatory conditions. Among these arenon-steroidal anti-inflammatory drugs (NSAIDS) and glucocorticoids. Amajor aspect of the mechanism of action of nonsteroidalanti-inflammatory drugs is generally thought to be the inhibition ofcyclooxygenase, the enzyme responsible for the biosynthesis of someprostaglandins and certain related autacoids. This inhibition isdependent upon the drug reaching the cyclooxygenase enzyme, indicatingthat the mode of action is at the level of interaction with the enzymeprotein itself. For example, acetaminophen can block the enzyme only inan environment that is low in peroxides which may explain its pooranti-inflammatory activity since sites of inflammation usually containhigh concentrations of peroxides generated by leukocytes. Aspirinacetylates a serine at or near the active site of cyclooxygenase,inhibiting the enzymatic activity. The most common unwanted side-effectof NSAIDS and other aspirin-like drugs is a propensity to induce gastricor intestinal ulceration. More serious side-effects, such as anemiafrom-resultant blood loss, may also sometimes occur.

Glucocorticoids have the capacity to prevent or suppress the developmentof the manifestations of inflammation. They are also of immense value intreating diseases that result from undesirable immune reactions. Theimmunosuppressive and anti-inflammatory actions of the glucocorticoidsare inextricably linked because they both result in large part frominhibition of specific functions of leukocytes, in particular,inhibition of lymphokines.

Two categories of toxic effects are observed in the therapeutic use ofadrenocorticosteroids⁷⁶ : those resulting from withdrawal and thoseresulting from continued use of large doses. Acute adrenal insufficiencyresults from too-rapid withdrawal of these drugs. Prolonged therapy withcorticosteroids may result in suppression of pituitary-adrenal functionthat can be slow in returning to normal. Further complications resultingfrom prolonged therapy with corticosteroids are: fluid and electrolytedisturbances; hypertension; hyperglycemia and glycosuria; increasedsusceptibility to infections; including tuberculosis; peptic ulcers,which may bleed or perforate; osteoporosis; a characteristic myopathy;behavioral disturbances; posterior subcapsular cataracts; arrest ofgrowth; and Cushing's habitus, consisting of "moon face", "buffalohump," enlargement of supraclavicular fat pads, "central obesity,"striae, ecchymoses, acne, and hirsutism.

An object of the present invention is to provide preparations of T.wilfordii Hook F, and isolated components thereof, for the treatment ofinflammation and for immunosuppression without steroidal agonist effect.Current NSAID treatment modalities are accompanied by significantlyundesirable side-effects. Therefore, agents that have theanti-inflammatory effects of NSAIDS without the side effects would be ofgreat benefit. A second object of the present invention is to suppressthe production of prostaglandins and other related autocoids withoutencountering the side effects of common NSAIDS. Accomplishment of theseobjectives will provide improved methods for treating inflammation, andimmunosuppression, such as in the treatment of autoimmune disease, andparticularly rheumatoid arthritis.

SUMMARY OF THE INVENTION

The present invention provides a method for treating inflammation or animmune disease in a subject while concurrently providing asteroid-sparing effect. These methods also limit, and in some casesessentially avoid, side effects associated with administration ofNSAIDS. The method comprises the step of administering to the subject apharmacologically active amount of a Tripterygium wilfordii Hook F rootpreparation to the subject, the preparation having anti-inflammatory andimmunosuppressive pharmacological activity.

The T. wilfordii purified preparation is obtained by a processcomprising extracting woody portions of Tripterygium wilfordii Hook Froot, in most embodiments skinless root or root wherein at least some ormost of the skin has been removed, to provide a preparation withreduced, or at least without substantial cellular toxicity, as comparedto crude preparations that have not been purified or otherwise extractedapart from contaminating components. The methods described as part ofthe present invention employ T. wilfordii preparations that are observedto have glucocorticoid receptor binding activity. Glucocorticoidreceptors are recognized by those of ordinary skill in the art asimportant in a number of pathologies. Thus, the methods are intended toencompass the use of these T. wilfordii components, in their isolatedand purified form, that demonstrate this unique glucocorticoid receptorbinding activity. Naturally derived and synthetically producedpreparations of said components of the T. wilfordii may therefore beefficaciously used in the practice of the described methods. Theglucocorticoid receptor binding active components of T. wilfordii havebeen characterized in root preparations, and may also exist in otherparts of the plant. Intended within the meaning of the glucocorticoidreceptor binding components of T. wilfordii are therefore thosecomponents, or pharmacologically active portions or fragments of thosecomponents, that bind glucocorticoid receptor. These components may befurther characterized as suppressing inflammation and immune responseswithout or with minimal steroidal agonist effects and/or without, orwith minimal NSAID effects.

The immune disease may be an autoimmune disease, such as rheumatoidarthritis, systemic lupus erythematosus, or psoriasis. The treatment ofinflammation according to the present methods provides inhibition of acyclooxygenase-2 dependent inflammatory process whereas the method ofimmunosuppression relates to suppression of interleukin-2 and ofinterferon γ production. Both of these effects relate to the capacity ofcomponents of TwHF to bind to the glucocorticoid receptor and inhibittranscription of genes with pro-inflammatory or immuno-enhancingactivity.

As used in the description of the present invention, the term "steroidsparing effect" is also defined as reducing the amount of steroid, suchas prednisone, capable of providing a beneficial pharmacological effectof the steroid. For example, upon treatment with the describedpreparations, patients receiving prednisone treatments may be maintainedon lower doses of the prednisone without significant loss of thepharmacological activity observed at the higher, initial doses of thesteroid. This phenomenon is exemplified in the data presented at Table13, in patients receiving the steroid prednisone. Thus, the"steroid-sparing effect" in the context of the present invention mayalso be described as the avoidance of commonly recognizedsteroid-related side effects, such as induction of glucose intolerance,osteoporosis, weight gain or a combination of these. This phenomenon mayalso relate to the absence of steroid agonist activity of a component(s)of the T. wilfordii preparation.

As also used in the description of the invention, the "woody" portion ofthe root is a portion of the root that is skinless, or without skin.Cellular toxicity as used in the present disclosure is measured by theinduction of cell death, interleukin-2 receptor expression, cellularsignaling activity, or a combination of one or more of these effects.Cellular signaling activity relates to inositol triphosphate production,diacylglycerol generation, translocation of protein kinase C, proteintyrosine kinase activity, or a combination of these activities.

An embodiment of the invention is a method for inhibitingcyclooxygenase-2 induction in a subject. The method comprisesadministering to the subject a pharmacologically active amount of aTripterygium wilfordii Hook F root preparation, or a pharmacologicallyactive component thereof, capable of binding glucocorticoid receptor. Inthis method, cyclooxygenase-1 activity is substantially unaffected.Inhibiting cyclooxygenase-2 induction inhibits the synthesis of aprostaglandin, an autacoid, or a cytokine inhibited by glucocorticoids,for example.

Constitutive production of prostaglandin and related autocoids iscatalyzed by cyclooxygenase-1. Cyclooxygenase is found in many cells.Inhibition of this enzyme has been associated at least in part with sideeffects observed with treatment that includes NSAIDS. Duringinflammation and after stimulation, inflammatory cells, such asmacrophage, transcribe a second gene and produce a new enzyme,cyclooxygenase-2, that accounts for much of the prostaglandin productionat inflammatory sites. NSAIDS also inhibit this enzyme. Steroids (andT2) inhibit transcription of the gene for cyclooxygenase-2 whilecyclooxygenase-1 activity is substantially unaffected. Therefore, theyhave the anti-inflammatory effects of NSAIDS but not the toxicity.

The cyclooxygenase-2 dependent inflammatory process may be the synthesisof a prostaglandin or the synthesis of a related autacoid. Theadditional corticosteroid-like immunosuppressive activities may be thesynthesis of a cytokine inhibited by glucocorticoids, such as IL-2 orinterferon γ, and may be related to a process of an autoimmune disease,such as, for example, rheumatoid arthritis, systemic lupus erythematosusor psoriasis.

Normally, when a glucocorticoid binds the glucocorticoid receptor, thebound complex induces the transcription of those genes activated by theglucocorticoid-receptor complex. The present inventors have shown hereinthat the TwHF extract, and components thereof, bind the glucocorticoidreceptor, but do not activate steroid sensitive genes. Consequently, thegenes that would normally be activated by that complex are notactivated. Therefore, the related undesirable effects of steroidtreatment are avoided by treatment with TwHF extract. Among the genesclearly shown by the present inventors to be inhibited by TwHF extractsare the IL-2 gene, the interferon γ and the cyclooxygenase-2 genes. Thisprovides a still further aspect of the invention as a method forinhibiting both immune reactions and inflammatory responses. Specificmethods for inhibiting glucocorticoid and receptor complexing with theTwHF extract preparations are also hereby disclosed. The components ofTwHF bind to the glucocorticoid receptor and that complex inhibitstranscription of certain genes such as IL-2, IFN γ and COX-2. However,unlike the complex of dexamethasone (a corticosteroid) and theglucocorticoid receptor, the complex of TwHF components and theglucocorticoid receptor does not activate genes that are driven byglucocorticoid response elements.

The undesired glucocorticoid receptor-dependent processes whoseactivation is avoided in the present invention may be induction ofglucose intolerance, osteoporosis, or weight gain, suppression ofpituitary-adrenal function, fluid or electrolyte disturbance,hypertension, hyperglycemia, glycosuria, susceptibility to infections,peptic ulcer, osteoporosis, myopathy, behavioral disturbance, posteriorsubcapsular cataracts, arrest of growth, or Cushing's habitus. In fact,all of the various hormone actions of glucocorticoids are driven byglucocorticoid response elements that regulate gene transcription.

The preparation may be a chloroform-methanol extract, achloroform-ethanol extract, an ethanol, or an ethyl acetate extract ofthe woody portion of the Tripterygium wilfordii Hook F root. Preferably,the extract is an ethyl acetate extract and, most preferably, theextract is obtained from an ethanol extraction followed by an ethylacetate extraction. The preparation may consist essentially oftripdiolide, triptolide, wilforonide, or related compounds of theextract that have the described biologic activity, or a compoundsynthesized having the basic triptolide structure that has triptolideactivity, and the pharmacologically active amount may be about 30-600mg/day, preferably about 50 mg/day to about 100 mg/day, or in someapplications, about 60 mg/day. However, more or less of any of thesedoses may be clinically appropriate depending on the needs and progressof the particular patient or condition being treated and the evaluationof the attending physician.

The pharmacologically active amount of the preparation of theTripterygium wilfordii Hook F root provided by the present invention isdemonstrated by its LD₅₀ in mice, which is greater than about 860 mg/kg.Expressed as a range, the LD₅₀ of the preparation is between about 860mg/kg to 1300 mg/kg. As demonstrated by the inventors, the EA extractproduced in Texas is demonstrated to have an LD₅₀, more preferably, ofabout 1250 mg/kg. The LD₅₀ of the preparation is much higher than thatobserved with other TwHF preparations, as shown herein, and therefore,the present preparation is considerably less toxic (i.e., requiringhigher doses to kill). This highlights the additional advantage of thepresent preparations as more pharmacologically acceptable.

The T. wilfordii preparation is further defined as having a therapeuticactivity:toxic index ratio greater than about 2.6×10⁻³, or preferably,from about 2.6×10⁻³ to 4.5×10⁻³ or more preferably, about 4.5×10⁻³. Thetherapeutic activity:toxic index ratio is calculated from an ID₅₀ invitro T-cell proliferation/LD₅₀ ratio.

The Tripterygium wilfordii Hook F preparation of the present method isalso described as having less than about 1.3 μg/mg triptolide orpreferably, about 0.2-1.3 μg/mg triptolide, or more preferably, about0.2 μg/mg triptolide. While the preparation may be obtained by any meansof chemical extraction techniques that yield a product having thedescribed therapeutic activity:toxic index ratio and triptolideconcentration, those techniques most preferred are ethyl acetateextraction of the root or by an ethanol extraction followed by an ethylacetate extraction of the root.

In a preferred embodiment of the above described method, the preparationof the Tripterygium wilfordii Hook F root extract is obtained by aprocess comprising the steps of i) obtaining woody portions of roots ofa Tripterygium wilfordii Hook F plant; and ii) extracting the woodyportions with a solvent to produce a Tripterygium wilfordii Hook Fpreparation, wherein the preparation has less than about 1.3 μg/mgtriptolide. The solvent is preferably ethyl acetate. Most preferably,the extracting step includes extracting with a first solvent and asecond solvent where the first solvent is ethanol and the second solventis ethyl acetate.

In a most preferred embodiment of the present invention, theTripterygium wilfordii Hook F preparation has a therapeuticactivity:toxic index ratio greater than about 2.6×10⁻³, and thepreparation is obtained by a process comprising the steps of i)obtaining woody portions of roots of a Tripterygium wilfordii Hook Fplant and removing the skin; ii) extracting the woody portions withethanol to produce an ethanol extract; and iii) extracting the ethanolextract with ethyl acetate to form a Tripterygium wilfordii Hook Fpreparation. The preparation has a therapeutic activity:toxic indexratio greater than about 2.6×10⁻³ and an LD₅₀ in mice of greater thanabout 860 mg/kg, and less than about 1.3 μg/mg triptolide. In thisembodiment of the present invention, the woody portions of the skinnedroots are dried to form a dried woody portion; the dried woody portionis ground to form a powder; and the powder is extracted with ethanol toproduce an ethanol extract; following the obtaining step aforedescribed.Most preferably, the woody portions of the root are to be dried underopen sunlight.

Methods for treating inflammation, an immune disease, or rheumatoidarthritis without substantially activating a steroid-dependent gene in apatient are a further embodiments of the present invention. The methodcomprises administering about 30 to about 600 mg/day of a Tripterygiumwilfordii Hook F root extract preparation having an LD₅₀ in mice ofgreater than about 860 mg/kg to the patient, wherein the preparationcontains less than about 1.3 μg/mg triptolide and wherein thepreparation binds to the glucocorticoid-receptor and thereby exertsanti-inflammatory and immunosuppressive activity.

A method for inhibiting glucocorticoid responsive genes, such as aninterleukin-2 gene, an interferon γ gene and a cyclooxygenase-2 gene, isa further aspect of the present invention. Each of these genes isdownregulated by a glucocorticoid receptor-binding molecular complex.Suppression of all pro-inflammatory or immune enhancing genes suppressedby glucocorticoids is envisioned by the present inventors. The methodcomprises the step of administering the above described preparation ofTripterygium wilfordii Hook F root extract in a pharmacologically activeamount. The pharmacologically active amount is as herein described.

A method of reducing progesterone activity comprising the administrationof the above described TwHF preparation is a further aspect of thepresent invention. The reduction of progesterone activity is useful forbirth control, for example, or as a "morning-after" treatment. Inaddition, this approach should be useful as an abortifacient.

A further embodiment of the present invention is a method fordetermining the ability of a candidate substance to bind theglucocorticoid receptor in a competitive binding assay in the presenceof TwHF preparation, or a glucocorticoid receptor binding componentthereof. The method includes generally the steps of admixing a candidatesubstance with a glucocorticoid receptor in the presence of TwHFpreparation or a glucocorticoid receptor binding component thereof, anddetermining binding of the candidate substance to the glucocorticoidreceptor. In this method, the glucocorticoid receptor may be from ahuman skin fibroblast preparation; the glucocorticoid receptor bindingcomponent may be triptolide, tripdiolide or wilforonide; theglucocorticoid receptor is preferably conjugated to a label, such as anenzymatic, chemical, or a radioactive label. A preferred label isavidin/biotin.

Where it is desired to select among candidate substances for thosewhich, like TwHF, do not activate steroid responsive genes, thecandidate substances that exhibit glucocorticoid receptor bindingactivity would further be screened to identify those which do notactivate steroid responsive gene expression. In so doing, othersubstances having the glucocorticoid receptor binding activity of TwHF,or its active components, that also avoid steroid-related side effects,may be identified.

A further embodiment of the invention is a method of selecting asubstance for treating inflammation or immune disease comprising thesteps of admixing a candidate substance with a glucocorticoid receptorin the presence of TwHF preparation or a glucocorticoid receptor bindingcomponent thereof, determining binding of the candidate substance to theglucocorticoid receptor, selecting a candidate substance having bindingaffinity for the glucocorticoid receptor, determining activity of theselected candidate substance for inducing steroid responsive geneexpression, and selecting the candidate substance being inactive forinducing steroid responsive gene expression. The second determining stepmay be carried out using a reporter gene construct under regulatorycontrol of a steroid responsive element, for example, the steroidresponsive element from the MMTV long terminal repeat. The reporter genemay be the luciferase gene, the chloramphenicol acetyltransferase gene,or the β-galactosidase gene, for example. One of skill in the art uponreading the present disclosure would know of similar reporter geneconstructs usable in the present invention.

Another embodiment of the invention is a method of blocking gammainterferon production in a subject. The method comprises administeringto the subject a pharmacologically active amount of a Tripterygiumwilfordii Hook F root preparation, or a pharmacologically activecomponent thereof, capable of blocking gamma interferon production. Amethod of inhibiting interleukin-2 gene transcription in a subject isalso an embodiment of the invention. The method comprises administeringto the subject a pharmacologically active amount of a Tripterygiumwilfordii Hook F root preparation, or a pharmacologically activecomponent thereof, capable of inhibiting interleukin-2 genetranscription.

Following long-standing convention of patent law practice, case law, andclaim construction, the words "a" and "an" denote "one or more", wherethey appear in the specification, including the claims.

The following abbreviations are used throughout the description of thepresent invention.

CRP=C reactive protein

DAG=diacylglycerol

ESR=erythrocyte sedimentation rate

FACS=fluorescence-activated cell sorter

GR=glucocorticoid receptor

Ig=immunoglobulin

IL-2=interleukin-2

IL-2R=interleukin-2 receptor

IP=phosphatidyl inositol triphosphate

MAb=monoclonal antibodies

MMTV=mouse mammary tumor virus

NHS=normal human serum

PBMC=peripheral blood mononuclear cells

PDB=phorbol dibutyrate

PHA=phytohemagglutinin

PMA=phorbol 12-myristate 13-acetate

PKC=protein kinase C RA

RA=rheumatoid arthritis

SA=formalinized Staphylococcus aureus

SK=streptokinase

SRBC=sheep red blood cells

T₂ =a chloroform/methanol extract from the woody portion of Tripterygiumwilfordii Hook F

TT=tetanus toxoid

TwHF or TWF=Tripterygium wilfordii Hook F

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Effect of T2 on T cell proliferation. T cells (1×10⁵ /well) werecultured with medium (□) or PHA (Δ) in the presence or absence ofvarying concentrations of T2 as indicated for 3 days. Results representthe mean cpm±SEM of three experiments.

FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E, FIG. 2F, FIG. 2G, FIG. 2H,FIG. 2I, FIG. 2J, FIG. 2K and FIG. 2L. Effect of T2 on cell cycleprogression of human T cells. T cells (1×10⁵ /well) were cultured withor without PHA (1 μg/ml) in the absence or presence of the indicatedconcentrations of T2 for 24, 48 or 72 hrs. The samples were harvested,stained with acridine orange, and analyzed with an ORTHO flow cytometerusing the CICERO program to determine the position of cells in the cellcycle as assessed by their RNA and DNA content.

FIG. 3. Inhibitory effect of T2 on IL-2 production. T cells (1×10⁵/well) were cultured with medium (□) or PHA (▪) in the presence orabsence of varying concentrations of T2 for 36 hrs. The cell-freesupernatants were diluted 1:4 and analyzed for IL-2 activity with CTLL-2cells. Mean ³ H!-TdR incorporation ±SEM of CTLL-2 cells from 6experiments is shown.

FIG. 4. Effect of supplemental IL-2 on T2 mediated inhibition of T cellproliferation. T cells (1×10⁵ /well) were stimulated with PHA with (□)or without (▪) IL-2 (10 U/ml) and in the presence or absence of varyingconcentrations of T2 for 3 days. The data are expressed as percent ofcontrol ³ H!-TdR incorporation from three experiments.

FIG. 5A and FIG. 5B. Effect of T2 on B cell DNA synthesis and Igproduction. In the left panel, B cells (5×10⁴ /well) were stimulatedwith SA (o) or SA+IL-2 (o), and in the right panel with SA+IL-2 in thepresence of varying concentrations of T2. ³ H!-TdR was determined aftera 5-day incubation (left panel). Supernatants were harvested after aseven-day culture and assayed for IgM (▪), IgG (o) and IgA (o) content(right panel). Results are the mean ±SEM of 3 experiments.

FIG. 6A and FIG. 6B. Effect of T2 on total IP generation by activated Tcells. Fresh T cells (A) or Jurkat cells (B) were labeled with ³H!-myo-inositol overnight in the absence or presence of the indicatedconcentrations of T2. Total IP was determined as described in Example 2.An aliquot of each cell population was also stimulated with PHA for 24hours and supernatants assayed for IL-2 content using CTLL-2 cells (o).Data are the mean of three replicate experiments.

FIG. 7A, FIG. 7B and FIG. 7C. Effect of T2 on the generation of IPfractions by PHA activated T cells. Jurkat cells were labeled with ³H!-myo-inositol overnight in the presence or absence of variousconcentrations of T2. Following a 5 minute incubation with 10 mM LiCl,the cells were activated with PHA for 60 min. Water soluble IPs wereisolated (IP1, top panel; IP2, middle panel and IP3, bottom panel) andquantitated as described in Example 2. Data are from one of threesimilar experiments.

FIG. 8. Effect of T2 on DAG generation and IL-2 secretion by PHAstimulated T cells. DAG and IL-2 were assayed as described in Example 2.Data represent the mean of duplicate determination of three similarexperiments.

FIG. 9A and FIG. 9B. Effect of T2 on translocation of PKC. PKC activityin both the cytoplasmic and membrane fractions were assayed as describedin Example 2.

FIG. 10 summarizes assessment of symptomatic improvement in rheumatoidarthritis patients as a result of treatment with a mixture fromTripterygium wilfordii Hook F.

FIG. 11 schematically shows the structure of triptolide (1) andtripdiolide (2).

FIG. 12 schematically shows the structure of triptonide.

FIG. 13 schematically describes the structure of wilfortrine (1) andwilfortrine methyl ester (2).

FIG. 14 shows the structure of triptophenolide (1) and triptophenolidemethyl ester (2).

FIG. 15 schematically shows the structure of triptonoterpenol.

FIG. 16 schematically shows the structure of wilformine.

FIG. 17 outline the extraction procedure for preparation of triptolide.

FIG. 18 depicts the structure of wilforonide (naphtho- 1,2-c!furan-3,7(1H,5H)-dione, 4,5a,6,8,9,9a-hexahydro-5a-methyl(5aR-trans)-104331-87-5!).

FIG. 19A and FIG. 19B show the elution profile of Fraction 924.

FIG. 20A, FIG. 20B, FIG. 20C and FIG. 20D show the function of Fraction924; 22A shows the effect of Fraction 924 on IL-2 production; 22B, theeffect on IL-2 expression; 22C, the effect on T-cell proliferation; and22D, the effect on cell viability.

FIG. 21 shows a comparison of the diterpenes in the ethyl acetateextracts of Tripterygium wilfordii Hook F prepared in China (CEA) andTexas (TEA). The diterpenes of each T. wilfordii Hook F preparation (CEAand TEA) were visualized by the Kedde reaction. Triptolide (3.6 μg),triptophenolide (10 μg) and tripdiolide (1.8 μg) served as referencestandards. The relative concentrations of triptophenolide, triptolideand tripdiolide of the CEA and TEA extracts are seen in this figure.

FIG. 22. TwHF inhibits binding of dexamethasone to human skin fibroblastglucocorticoid receptor (GR).

FIG. 23. TwHF inhibits GR-mediated target gene activation. The targetgene was the luciferase gene under regulatory control ofglucocorticoid-inducible elements in the MMTV long terminal repeat.

FIG. 24. Dose response curves of TwHF effect on GR ligand binding andtarget gene activation. Data of FIGS. 20 and 21 are replotted togetheras a maximal binding or activity as a function of log TwHFconcentration.

FIG. 25A, FIG. 25B and FIG. 25C. The extract of TwHF and purifiedcomponents inhibit dexamethasone dependent growth of IDH4 cells in aconcentration dependent manner. IDH4 cell proliferation was assessed by³ H-thymidine incorporation after a 72 hour culture. (The n in 10^(n) isthe abscissa of the graph, i.e., 1, 2, 3, 4, or 5; indicating nanomolarconcentration).

--□--Dex 1,000 nM

--♦--Dex 100 nM

--▪--Dex 10 nM

--⋄--Dex 1 nM

FIG. 26. Cortisol RIA with a novel A ring directed anti-cortisolantibody detects immunoreactivity in TwHF extract. Data are expressed asthe fraction of radioactive cortisol tracer bound to antibody (B/B_(o))as a function of added cortisol or TwHF. The TwHF displacement curve isnot parallel to that of authentic cortisol, but some cross reactivitymay exist.

FIG. 27. Dose response curves for the TwHF compound A effect on GRligand binding activity (as for FIG. 22) and GR-mediated target geneactivation (as for FIG. 23). Data are shown as % maximal binding oractivity as a function of TwHF concentration (log scale).

FIG. 28. Cortisol RIA with A ring-directed antibody fails to detectcortisol-like immunoreactivity in TwHF purified compounds A and B(triptolide and tripdiolide, respectively). Compound C (triptophenolide)may have low affinity immunoreactivity.

FIG. 29A, FIG. 29B, FIG. 29C and FIG. 29D. The extract of TwHF andpurified components inhibit endotoxin induced PGE₂ production by humanperipheral blood monocytes, RU 486 is tested in FIG. 29D.

FIG. 30. Chromatogram of triptolide and acetophenone. Peaks:1=acetophenone; 2=triptolide. Conditions: Nova-Pak C18 stainless steelcolumn (150 mm×3.9 mm I.D.); mobile phase, acetonitrile-water (19:81);flow-rate, 1.5 ml/min.; UV monitor at 214 nm; sample volume 10 ul.

FIG. 31. Chromatogram of tripdiolide. Peak: 1=tripdiolide. Mobile phaseacetonitrile-water (11:89); flow-rate, 2.0 mL/min. Other conditions asin FIG. 30.

FIG. 32. Chromatogram of the extract of Tripterygium wilfordii Hook F.with the internal standard for the determination of triptolide. Peak:1=acetophenone; 2=triptolide. Conditions as in FIG. 30.

FIG. 33. Chromatogram of the extract of Tripterygium wilfordii Hook F.for the determination of tripdiolide. Peak 1=tripdiolide. Conditions asin FIG. 31.

FIG. 34 shows the effect of the EA extract on the production of IL-2 andIFN-γ. T cells (1×10⁵ /ml) were incubated with PHA (1 μg/ml) for 24 hrin the presence or absence of the indicated concentrations of the EAextract. --□--, IFN-γ, --♦--, IL-2.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention concerns the use of Tripterygium wilfordii Hook Fextracts, and components thereof, to inhibit inflammatory processes andimmune disorders concurrently with the induction ofglucocorticoid-receptor-mediated inhibition of expression ofpro-inflammatory genes. The inhibition of the activation of thesepro-inflammatory and immune enhancing genes provides a treatment forinflammation or an immune disorder. The further demonstration that thesecomponents of TwHF lack steroidal agonist effects indicates that thismethod may therefore be used to treat autoimmune disease, such asrheumatoid arthritis, to suppress immune function and to suppressinflammation without the undesirable effects of steroids or NSAIDs thatare in current use.

Methods for binding to the glucocorticoid receptor are also provided,demonstrating that coupling of glucocorticoid to the glucocorticoidreceptor is inhibited in the present of the TwHF preparation. Anattractive alternative to use of steroid containing therapeutics intreatment of inflammation and other disorders is thus provided, use ofTwHF avoiding the various steroid-related side effects associated withthese agents, as well as avoiding the gastric irritation associated withNSAIDs. Therapeutic agents for the treatment and prevention ofinflammation that are NSAID and steroid-free are also provided, thesepreparations containing as an active ingredient a pharmaceuticallyeffective amount of a TwHF extract. Because the TwHF extract providesanti-inflammatory activity, amounts of other agents such as an NSAID ora steroid can be reduced to amounts better tolerated by the patientwhile providing an alternative medicinal function, i.e., such as thefever reducing or pain relieving function of NSAIDs.

The extract of TwHF inhibits the induction of Cyclooxygenase-2 (COX-2)but has no direct effect on cyclooxygenase-1 (COX-1) and probably has nodirect effect on the enzymatic activity of either. Therefore, thepresent invention provides for the blocking of inflammation by blockingthe induction of COX-2, leaving COX-1 intact and thereby causing none ofthe side-effects of NSAIDS that are related to COX-1 inhibition.

T2 was prepared by chloroform-methanol and chloroformethanol extractionof Tripterygium wilfordii Hook F roots, the skin of the roots havingbeen removed prior to processing the root. As an additional example, thepreparation is prepared by ethyl acetate extraction (Example 5).

Example 1 concerns studies on the effect of the Tripterygium wilfordiiHook F preparation on human lymphocyte function. Interleukin-2production by T cells is inhibited by the TwHF extract by inhibitinggene transcription, while the expression of IL-2 receptors is notaffected. The Tripterygium wilfordii Hook F preparation is also shown tosuppress proliferation of B cells and immunoglobulin production by Bcells. The studies described in Example 2 indicate that signalingpathways are not affected by the Tripterygium wilfordii Hook Fpreparation, demonstrating the selective nature of the agent'sinhibitory activity. Example 3 concerns studies on the effect of the T.wilfordii preparation in the treatment of patients with rheumatoidarthritis. Example 4 describes the components of the T₂ T. wilfordiiHook F preparation and toxicity thereof. Example 5 describes the T.wilfordii Hook F preparation obtained by ethyl acetate extraction, whileExample 6 demonstrates the in cellulo (intact cell effects) activity ofthe ethyl acetate extract.

Example 7 provides in vivo studies with the T. wilfordii ethyl acetateextract. Example 8 provides a comparative toxicity study of various T.wilfordii preparations. Example 9 describes a technique that may be usedfor determining the dose schedule of the ethyl acetate extract for usein vivo. Example 10 describes the characterization and identification ofa fraction `924` (wilforonide) from the ethyl acetate extract andExample 11 describes immunosuppressive effects of wilforonide. Example12 provides studies that show inhibition of IL-2 gene transcription andinhibition of production of interferon γ by the T2 extract. Example 13demonstrates inhibition of glucocorticoid-receptor-mediated activationof gene transcription concurrently with a lack of intrinsic GR agonistactivity. The significance of these results is that inflammation may beaddressed in a subject using T2 without steroids, thus avoiding theundesired agonist effects associated with steroids. Example 14 providesstudies of the anti-inflammatory properties of the TwHF extract. A newmethod for separating and quantitating triptolide and tripdiolide isprovided in Example 15 and Example 16 describes the effect of TwHFextract on progesterone metabolism, highlighting the utility of thepresent invention for reducing progesterone, and the variousphysiological conditions that are related to progesterone (i.e.,pregnancy). Example 17 provides therapeutic preparations of the TwHFextract.

EXAMPLE 1 Effect of T₂ on Human Lymphocyte Function

This example describes the effect of the T. wilfordii preparation, T₂,on in vitro immune responsiveness of human peripheral blood mononuclearcells (PBMC) obtained from normal individuals. It was found that thepreparation exerted a concentration-dependent profile of suppressiveactivity on both T cell and B cell functions, whereas the functionalactivities of monocytes were more resistant to the suppressive effectsof this T. wilfordii preparation with chloroform/methanol.

Methods

Cell preparation. PBMC were obtained from the blood of healthy adults bycentrifugation on sodium diatrizoate/Ficoll gradients (Sigma, St. Louis,Mo.). Monocytes were isolated from PBMC by centrifugation onSepra-cell-MN (Sepratech, Oklahoma City, Okla.) or by glass adherence.The monocytes obtained from the two procedures were used to examineinterleukin-1 (IL-1) production and antigen presentation, respectively.For purification of T cells and B cells, PBMC were incubated withL-leucine methyl ester HCl (Sigma) for 45 minutes at room temperature todeplete monocytes and natural killer cells⁵¹. The resultant lymphocyteswere rosetted with neuraminidase-treated sheep red blood cells (SRBC)and were then separated by Ficoll/diatrizoate centrifugation⁵². T cellswere further purified by passage of the rosette-positive population overa nylon-wool column to remove residual B cells and monocytes⁵³. B cellswere prepared from the initial population of rosette-negative cells byremoving any remaining cells that formed rosettes withneuraminidase-treated SRBC.

Staining with monoclonal antibodies (MAb) to CD3 and CD20 and analysiswith the fluorescence-activated cell sorter (FACS) indicated that the Tcell and B cell populations were more than 96% and 90% pure,respectively. T cells were incubated with mitomycin c (0.1 mg/ml) for 45minutes and then washed thoroughly⁵⁴.

Reagents, The T. wilfordii preparation, T₂, used in these studies was achloroform/methanol extract prepared from the woody portion of the rootsof TWH obtained from Taizhou Pharmaceutical Company (Taizhou, Jiang Su,People's Republic of China). This preparation, T₂, contained more than 8different compounds including glycosides, diterpenoids, alkaloids, andketones. Before use, the extract was dissolved in DMSO and furtherdiluted with culture medium. Phytohemagglutinin (PHA; Wellcome Reagents,Research Triangle Park, N.C.), phorbol dibutyrate (PDB; Sigma),ionomycin (Calbiochem, San Diego, Calif.), and the anti-CD3 MAb, 64.1,were used for T cell activation⁵⁵. MAb 64.1 was purified as previouslydescribed Hansen et al., "T cell protocol", Leukocyte Typing. Edited byBernard, et al. Berlin, Springer-Verlag, 1982!. Human recombinantinterleukin-2 (rIL-2; Cetus, Emeryville, Calif.) and/or formalinizedStaphylococcus aureus (SA; Calbiochem) was used for B cell activation.The MAb against the α chain of the IL-2 receptor (IL-2R), anti-Tac, wasobtained from Dr. Thomas Waldmann (NIH, Bethesda, Md.) and was used toanalyze IL-2R expression. Interleukin-1 (Cistron Technology, Pine Brook,N.J.) was purchased for standardization of the IL-1 assay.Affinity-purified goat anti-human IgA, IgG, and IgM and similarantibodies conjugated to horseradish peroxidase were purchased from Tago(Burlingame, Calif.). Streptokinase (SK) and tetanus toxoid (TT) werepurchased from Hoechst-Roussel (Somerville, N.J.) and MCDC Biologics(Jamaica Plain, Mass.), respectively.

Cell culture and assay of lymphocyte DNA synthesis. T cells (1×10⁵/well) or B cells (5×10⁴ /well) alone or B cells with mitomycinc-treated T cells (1×10⁵ /well) were cultured in RPMI 1640 medium(Hazleton Biologics, Lenexa, Kans.) supplemented with 10% fetal calfserum, penicillin G (200 units/ml), gentamicin (10 μg/ml), andL-glutamine (0.3 mg/ml) in 96-well microtiter plates in a total volumeof 200 μl, with or without the stimuli indicated, and in the presence orabsence of various concentrations of T₂. The final concentration of DMSOin culture was 0.02-0.002%. This concentration of DMSO had no effect onany of the responses analyzed.

For both T and B cell activation, immobilized anti-CD3 (MAb 64.1)stimulation was used. This MAb was immobilized by incubating 50 μl (5μg/ml) in each well for at least 2 hours at room temperature. The excesssoluble antibody was removed before cell culture (Hansen, supra). Cellswere cultured for the indicated duration, and then pulsed with 1 μCi of³ H-thymidine, (³ H-TdR; New England Nuclear, Boston, Mass.) for thelast 12 and 18 hours for T cell and B cell cultures, respectively. ³H-TdR uptake was measured in a liquid scintillation counter. All dataare expressed as the mean counts per minute of 3 replicatedeterminations⁵⁶.

IL-1 production assay. Monocytes (1×10⁵ /well) were suspended in RPMI1640 medium with 1% normal human serum (NHS) and cultured with orwithout lipopolysaccharide (10 μg/ml) in the presence or absence ofvarious concentrations of T₂ for 24 hours. The culture supernatants werecollected, and serial dilutions were assayed for IL-1 using C3H/HeJmurine thymocytes as described elsewhere⁵⁷. Concentrations of T₂contained in the dilutions of supernatants had no effect on DNAsynthesis by C3H/HeJ thymocytes.

IL-2 production assay. T cells (1×10⁵ /well) were incubated with orwithout PHA (1 μg/ml) or immobilized anti-CD3 in the presence or absenceof various concentrations of T₂ for 24 hours. Cell-free supernatantswere harvested, serial dilutions were made, and IL-2 content was assayedwith CTLL-2 cells as described previously⁵⁸.

IL-2R expression. T cells were cultured with or without the indicatedstimuli in the presence or absence of various concentrations of T₂ for36 hours. After washing, the cells were stained with saturatingconcentrations of anti-Tac or a mouse IgG control MAb, followed byfluorescein isothiocyanate-conjugated goat anti-mouse Ig antibody(Cappel, West Chester, Pa.). The samples were fixed with 1%paraformaldehyde and analyzed with a FACSTAR (Becton Dickinson, MountainView, Calif.) flow cytometer, using a single-histogram statisticsprogram (Davis, supra).

Measurement of Ig synthesis. The amount of IgG, IgA, and IgM in theculture supernatants of B cells stimulated with SA plus rIL-2 in thepresence or absence of T₂ for 7 days was determined using anisotype-specific enzyme-linked immunosorbent assay method. Quantitationof the Ig in the supernatants was then determined by comparison with astandard curve. The sensitivity of the assay is 15 ng/ml for IgA andIgG, and 30 ng/ml for IgM⁵⁹.

Results

Effect of T₂ on human T cell responsiveness. These studies demonstratethat T₂ caused concentration dependent inhibition of PHA induced ³H-thymidine incorporation by purified human T lymphocytes (FIG. 1).Fifty percent inhibition was noted at concentrations of approximately0.2 μg per ml. Cell cycle analysis indicated that T₂ prevented cellsfrom progressing through the G1 phase of the cell cycle (FIGS. 2A-2L).Mitogen induced IL-2 production by purified T-cells was also inhibitedby a similar concentration of T₂ (FIG. 3). Mitogen induced expression ofIL-2 receptors was not inhibited by T₂ (Table 1) indicating that it wasnontoxic to this cellular activity. These results suggested that thedecrease in proliferation might be the result of inhibition of IL-2production.

                  TABLE 1                                                         ______________________________________                                        EFFECT OF T.sub.2 ON INTERLEUKIN-2 (IL-2)                                     RECEPTOR EXPRESSION*                                                          Nil                      PHA                                                                   Fluorescence       Fluorescence                              T.sub.2 % positive                                                                             intensity   % positive                                                                           intensity                                 ______________________________________                                        0 g/ml  10 ± 2                                                                              483 ± 18 65 ± 17                                                                           561 ± 166                              0.65 μg/ml                                                                         --       --          60 ± 22                                                                           519 ± 109                              1.25 μg/ml                                                                          9 ± 2                                                                              504 ± 29 61 ± 20                                                                           525 ± 128                              2.50 μg/ml                                                                         --       --          --     --                                        ______________________________________                                         *T cells (1 × 10.sup.5 /well) were cultured with medium or              phytohemagglutinin (PHA) in the presence or absence of various                concentrations of T.sub.2 as indicated for 36 hours. Cells were collected     stained with antiTac monoclonal antibody followed by fluorescein              isothiocyanateconjugated goat antimouse IgG, and analyzed by flow             cytometry. Values are the mean ± SEM of 6 experiments.                

In order to examine IL-2 production, experiments were carried out inwhich the effect of T₂ on proliferation was examined in the presence ofsupplemental IL-2. As can be seen in FIG. 4, much of the inhibitoryeffect of T₂ was overcome by supplemental IL-2. The effect of T2 onsteady state levels of IL-2 mRNA in mitogen stimulated T cells wasstudied. T cells (1×10⁶ /ml) were cultured with and without PHA in thepresence or absence of T2 (1 μg/ml). After a 4-hour incubation, totalRNA was isolated and IL-2 and actin mRNA levels determined by S1nuclease protection. The results suggested that one of the major actionsof T₂ was to inhibit IL-2 production. This appeared to result from aninhibition of IL-2 gene transcription since T₂ inhibited the appearanceof mRNA for IL-2. These experiments confirmed that one action of T₂ wasto inhibit IL-2 production.

A method of testing for selective inhibition of IL-2 specific mRNAproduction is described herein, the method consists of: culturingeukaryotic cells in culture with and separately without Tripterygiumwilfordii Hook F T₂ extract or components thereof in a therapeuticallyeffective amount to provide a test sample and a control sample;measuring IL-2 mRNA level and a reference MRNA level such as actin MRNAto provide a test IL-2 mRNA sample, a test reference mRNA sample, acontrol IL-2 mRNA sample and a control reference mRNA sample; comparing(test IL-2 MRNA level÷control IL-2 mRNA level) to (test reference mRNAlevel÷control reference mRNA level); and when (test IL-2 mRNAlevel÷control IL-2 MRNA level) is substantially less than 1 and (testreference mRNA level÷control reference mRNA level) is about 1, selectiveinhibition of IL-2 mRNA production by T₂ is indicated.

Effect of T₂ on human B lymphocyte responses.

Additional effects of T₂ were demonstrated when its action on human Bcell responses was examined. As can be seen in FIGS. 5A and 5B, T₂inhibited both mitogen-induced proliferation of highly purified B cells,as well as immunoglobulin production in a concentration dependentmanner. These results suggested that T₂ had additional effects beyondaltering IL-2 production. Some specificity for the action of T₂ wasdemonstrated, however, when its effects on a number of other cell typeswere examined. Thus, T₂ had no effect on IL-1 production by humanmonocytes nor on their capacity to function as antigen presenting cells.In addition, there was no effect on the growth of endothelial cells orfibroblasts during a 48 hour culture. None of the inhibitory effects ofT₂ could be accounted for by non-specific toxicity, as inhibitoryconcentrations of T₂ had no effect on the viability of either resting orstimulated lymphocytes, endothelial cells, fibroblasts, monocytes, orpolymorphonuclear leukocytes. These results support the contention thatT₂ has a limited spectrum of immunosuppressive activity which cannot beaccounted for by nonspecific toxic effects. Of importance, the capacityof T₂ to suppress both IL-2 production by T cells, and proliferation andimmunoglobulin production by B cells may explain the action of thisagent in patients with RA.

EXAMPLE 2 Effect of T₂ on Critical Signaling Pathways

The mechanism by which T₂ inhibits IL-2 production is examined ingreater detail in the present example. T. wilfordii may inhibit acritical signaling pathway involved in inducing transcription of theIL-2 gene. Current information suggests that T cell receptor occupancyleads to activation of tyrosine kinases, followed by stimulation ofphospholipase C. This results in production of phosphatidyl inositoltriphosphate and diacylglycerol, that induce increases in intracellularcalcium and activation of protein kinase C, respectively⁶⁰. Therefore,additional studies were carried out to examine the possibility that T₂might inhibit one of these signaling pathways.

Methods

Effect of T₂ preparation on total IP generation by activated T cells.Fresh T cells (A) or Jurkat cells (B) were labeled with ³H!-myo-inositol overnight in the absence or presence of the indicatedconcentrations of the T. wilfordii preparation obtained bychloroform/methanol extraction was obtained from the supplier sourcedescribed in Example 1. The cells were washed and incubated with 10 mMLiCl for 5 minutes then activated with PHA for 60 min. The cells wereextracted with 0.75 ml of a 1:1 mixture of chloroform and methanol,followed by 0.25 ml of chloroform and 0.25 ml of water. The phases wereseparated by centrifugation and the water soluble fractions were appliedto a 0.25 ml Agl-X8 formate ion exchange column. Total inositolphosphate was eluted with 1.5 ml of 0.1M formic acid and 1M sodiumformate. The radioactivity was quantified by scintillation counting. Analiquot of each cell population was also stimulated with PHA for 24hours and supernatants assayed for IL-2 content using CTLL-2 cells.

Effect of T₂ on the generation of IP fractions by PHA activated T cells.Jurkat cells were labeled with ³ H!-myoinositol overnight in thepresence or absence of various concentrations of T₂. Following a 5minute incubation with 10 mM LiCl, the cells were activated with PHA for60 min. Water soluble IPs were isolated and quantitated. To accomplishthis, the cultures were extracted with 0.75 ml of a 1:1 mixture ofchloroform/methanol, followed by 0.25 ml each of chloroform and water.The phases were separated by centrifugation and the water solublefraction was applied to a 0.25 ml Agl-X8 formate ion-exchange column,and washed extensively with 5 mM cold myoinositol. IP1, IP2 and IP3 weresequentially eluted with 4 ml of 0.2M ammonium formate plus 0.1M formicacid, 10 ml of 0.4M ammonium formate plus 0.1M formic acid and 10 ml of1M ammonium formate plus 0.1M formic acid respectively. Theradioactivity of the various elution fractions was quantified byscintillation counting.

Effect of T₂ on DAG generation and IL-2 secretion by PHA stimulated Tcells. T cells for each sample were cultured overnight with PHA in thepresence or absence of the indicated concentrations of T₂. The cellpellets were lysed with a mixture of chloroform and methanol, andfractions separated with 1M NaCl and chloroform. The organic phase wascollected and dried under nitrogen. DAG mass in the organic extract wasassayed by solubilizing the lipid residues in a mixture of ³² P-γ-ATPand DAG kinase and phosphatidic acid, incubating at 37° C. for 1 hourduring which DAG was quantitatively converted to p-phosphatidic acid.The samples were dried and redissolved in chloroform. The solvent wasapplied to a silica gel and separated by thin layer chromatography withchloroform/methanol/acetic acid. After visualization with iodine, thespot which contained phosphatidic acid was harvested and radioactivitydetermined by liquid scintillation counting. An aliquot of cells wasalso stimulated with mitogen and supernatants harvested after 24 hoursand assayed for IL-2 content.

Effect of T₂ on translocation of PKC. Jurkat cells (1×10⁶ /ml) wereincubated overnight with or without T₂ at the indicated concentrations.The cells were lysed by sonication and then cytoplasmic and membranefractions separated by centrifugation. PKC activity in both thecytoplasmic and membrane fraction was assayed using a protein kinase Cassay system (Amersham) which employed a synthetic peptide as aphosphate acceptor in the presence of phosphatidylserine, calcium andPMA.

Effect of T₂ on protein tyrosine phosphorylation. Jurkat cells (3×10⁶)were incubated overnight in the absence or presence of the indicatedconcentrations of T₂. The cells were washed and stimulated with PHA for30 minutes. After centrifugation, the pelleted cells were solubilizedwith 1×SDS sample buffer containing protease inhibitors. The lysateswere centrifuged at 10,000 rpm for 15 minutes. The supernatants wereanalyzed for protein phosphorylation by western blotting using a mousemonoclonal antibody (Upstate Biotechnology, Inc.) againstphosphotyrosine.

Results

The effect of T₂ on mitogen induced production of phosphatidyl inositolmetabolites. As can be seen in FIGS. 6A-6B, mitogenic stimulation leadto the production of IL-2 and phosphatidyl inositol metabolites. WhereasIL-2 production was inhibited, generation of phosphatidyl inositolmetabolites was not. Similar results were seen in fresh T cells and inthe Jurkat leukemic T cell line. Additional studies examined whether T₂specifically inhibited generation of IP3, which is thought to induceincreases in intracellular calcium⁵². As can be seen in FIGS. 7A-7C, T₂had no effect on the generation of IP3 or other specific PI metabolitesby mitogen activated T cells. Similar experiments examined the effect ofT₂ on the generation of diacylglycerol. As can be seen in FIG. 8, T₂inhibited IL-2 production from mitogen stimulated T cells, but had noeffect on DAG production. Additional of the inventors studies, notshown, examined the activity of T₂ on phospholipase C activity isolatedfrom fresh T cells or Jurkat cells. Again, no inhibitory activity wasobserved. These experiments suggested that the action of T₂ cannot beexplained by an effect on these early signaling pathways. At theselevels of T₂ extract addition, nontoxicity to other cellular functionsis established as indicated by these cellular assays.

The effect of T₂ on protein kinase C activation. As can be seen in FIGS.9A-9B, mitogen stimulation led to translocation of PKC in Jurkat cells,and T₂ did not effect PKC translocation. Finally, the effect of T₂ onthe activity of protein tyrosine kinase activity was explored. Mitogenicstimulation of T cells lead to phosphorylation of a number of proteinspecies identified with a specific antibody to phosphotyrosine. However,T₂ did not inhibit the activity of protein tyrosine kinase since thesame bands were observed regardless of the presence of T₂ duringmitogenic stimulation. These experiments convincingly demonstrate thatT₂ has no effect on early signaling pathways involved in induction ofIL-2 gene transcription.

EXAMPLE 3 In Vivo Trials

In an open trial, it was found that a mixture of compounds (T₂)extracted from Tripterygium wilfordii Hook F was effective in thetreatment of rheumatoid arthritis.

To confirm the previous results obtained from these open studies, aprospective, controlled, double-blind cross-over study was designed andcarried out.

The treatment plan was designed as follows:

Seventy patients with classic or definite adult-onset rheumatoidarthritis who had active disease for more than 6 months were acceptedinto the trial and randomly assigned to 2 treatment groups. Patients inGroup A received T₂ for a first course of treatment of 12 weeks, andthen were subsequently changed to placebo for a second course oftreatment of 4 weeks duration. Patients of Group B received placeboduring the first course and then were crossed-over and received T₂therapy during the second course. T₂ was taken in a dosage of 60 mgdaily. Placebo tablets were identical in appearance to T₂ tablets. Table2 shows the treatment plan schedule.

                  TABLE 2                                                         ______________________________________                                        TREATMENT PLAN (TOTAL COURSE: 16 WEEKS)                                               First course treatment                                                                     Second course treatment                                          (12 weeks)   (4 weeks)                                                ______________________________________                                        Group A   T.sub.2, 20 mg t.i.d.                                                                        Placebo                                              Group B   Placebo        T.sub.2, 20 mg t.i.d.                                ______________________________________                                    

All patients were assessed in an arthritis clinic every 4 weeks. Theclinical assessment, overall assessment by physicians and drugdistribution were carried out by individual doctors in a blinded manner.The laboratory assessments were done by technicians of a centralhospital laboratory, who were also blinded to the details of the trial.

                  TABLE 3                                                         ______________________________________                                        CLINICAL FEATURES OF PATIENTS ENTERING THE TRIAL                                                    Second                                                            First Treatment Course                                                                    Treatment Course                                                  Group A                                                                              Group B  Group A   Group B                                             T.sub.2                                                                              Placebo  Placebo   T.sub.2                                   ______________________________________                                        Number of Patients                                                                        35       35       27      31                                      Male/Female 3/32     4/31     1/26    4/27                                    Mean age, years                                                                           46.3     48.0     46.2    47.7                                    Mean disease duration                                                                     5.9      6.1      5.8     6.0                                     (years)                                                                       Stage of Disease                                                              (1)         6        6        4       5                                       (2)         14       16       11      13                                      (3)         12       10       10      9                                       (4)         3        3        2       4                                       ______________________________________                                    

The clinical features of patients entering the trial are shown in Table3. Statistical analyses demonstrated that at the beginning of the trial,Group A and Group B did not differ from each other significantly in age,sex, duration of disease or stage of disease.

                  TABLE 4                                                         ______________________________________                                        RESULTS OF A CONTROLLED TRIAL                                                 OF T.sub.2 IN RHEUMATOID ARTHRITIS                                                             No. of Patients                                                               Completing Treatment                                                    No. Beginning                                                                             First Course                                                                            Second Course                                Gruop      Treatment   (12 wks)  (4 wks)                                      ______________________________________                                        A (T.sub.2 -> Placebo)                                                                   35          27        24                                           B (Placebo -> T.sub.2)                                                                   35          31        25                                           ______________________________________                                    

As shown in Table 4, 27 patients of Group A completed the first courseof treatment, of which 24 completed the second course. 31 and 25 ofGroup B completed the first course and second course of treatment,respectively.

Table 5 indicates the reasons patients withdrew from the study. Threepatients of Group B but none of Group A withdrew from the trial becauseof worsening of disease during the first course of treatment, whereas 4patients from Group A but none from Group B withdrew from the trialbecause of side effects.

                  TABLE 5                                                         ______________________________________                                        REASONS FOR WITHDRAWAL FROM THE STUDY                                                 First Course Treatment                                                                    Second Course Treatment                                           Group A                                                                              Group B  Group A   Group B                                             T.sub.2                                                                              Placebo  Placebo   T.sub.2                                             (n = 5)                                                                              (n = 35) (n = 27)  (n = 1)                                             No.  %     No.    %   No.  %    No.   %                               ______________________________________                                        Lost to   4      11    1    3   3    11   6     19                            follow up                                                                     Worsening of                                                                            0      0     3    9   0    0    0     0                             disease                                                                       Side effects                                                                            4      11    0    0   0    0    0     0                             ______________________________________                                    

Table 6 shows the therapeutic effects of the first course of treatment.In comparison with patients of Group B, patients of Group A showedsignificant improvement in all clinical assessments including morningstiffness, joint tenderness score, number of swollen joints, gripstrength and 15 meter walking time.

                  TABLE 6                                                         ______________________________________                                        CHANGES IN CLINICAL PARAMETERS IN PATIENTS                                    COMPLETING THE FIRST COURSE OF TREATMENT                                                     Group A Group B                                                               T.sub.2 Placebo                                                               (n = 27)                                                                              (n = 31)  *p                                           ______________________________________                                        Morning stiffness                                                                         Before   2.4 ± 0.4                                                                            1.1 ± 0.2                                   (hours)     After    0.9 ± 0.2                                                                            2.3 ± 1.4                                                                          0.01                                   Joint tenderness                                                                          Before   25.1 ± 1.9                                                                           25.5 ± 1.7                                  score       After    7.9 ± 1.3                                                                            21.9 ± 2.1                                                                         0.001                                  Number of swollen                                                                         Before   9.2 ± 0.9                                                                            7.8 ± 0.7                                   joints      After    4.3 ± 0.6                                                                            7.4 ± 1.1                                                                          0.01                                   Grip strength                                                                             Before   49.0 ± 0.4                                                                           73.6 ± 7.7                                  (mean of both                                                                             After    84.4 ± 7.5                                                                           81.2 ± 8.9                                                                         0.05                                   sides, mm Hg)                                                                 15 meter walking                                                                          Before   36.6. ± 6.6                                                                          37.0 ± 2.4                                  time (second)                                                                             After    21.6 ± 1.5                                                                           31.9 ± 3.6                                                                         0.05                                   ______________________________________                                    

The most noteworthy improvement was observed in joint tenderness score,which improved from a mean of 25.1 before entry to a mean of 7.9 afterthe first course of treatment with T₂. By contrast, there were nosignificant changes in this score in Group B patients treated withplacebo.

As shown in Table 7, treatment with T₂ also caused improvement inlaboratory correlates of disease activity. Significant improvements inESR, CRP and immunglobulin levels were noted. The changes weresignificant at the p 0.001 level when compared between Group A and GroupB.

                  TABLE 7                                                         ______________________________________                                        CHANGES IN LABORATORY PARAMETERS IN PATIENTS                                  COMPLETING THE FIRST COURSE OF TREATMENT                                                   Group A  Group B                                                              T.sub.2  Placebo                                                              (n = 27) (n = 31)   *p                                           ______________________________________                                        ESR (mm/hour)                                                                           Before   69.2 ± 6.4                                                                            63.9 ± 5.2                                             After    41.0 ± 5.9                                                                            67.2 ± 6.6                                                                          <0.001                                 CRP (u/ml)                                                                              Before   29.4 ± 5.7                                                                            31.6 ± 4.1                                             After    10.4 ± 3.9                                                                            43.7 ± 7.0                                                                          <0.001                                 RF (titers)                                                                             Before   87.1 ± 23.2                                                                           86.1 ± 35.5                                            After    48.0 ± 13.4                                                                           63.4 ± 10.9                                                                         NS                                     IgG (μ/ml)                                                                           Before   227.5 ± 4.6                                                                           231.9 ± 14.2                                           After    117.4 ± 9.5                                                                           180.4 ± 29.8                                                                        <0.001                                 IgM (μ/ml)                                                                           Before   302.8 ± 40.3                                                                          284.5 ± 32.2                                           After    105.2 ± 11.1                                                                          261.3 ± 29.3                                                                        <0.001                                 IgA (μ/ml)                                                                           Before   289.6 ± 29.4                                                                          257.6 ± 25.2                                           After    149.0 ± 15.5                                                                          280.4 ± 29.8                                                                        <0.001                                 ______________________________________                                         *Group A vs Group B                                                      

There was a greater tendency to decrease RF titer in T₂ treated patientsbut the difference between the two groups after the first course oftreatment was not statistically significant.

During the second course of therapy, patients who had received placeboinitially improved significantly after 4 weeks of therapy with T₂. (SeeTable 8). Significant improvements in joint tenderness score, number ofswollen joints and grip strength were noted. Improvement in morningstiffness and 15 meter walking time were also noted, but these changesdid not achieve statistical significance. Patients who had received T₂during the first 12 weeks of therapy continued to maintain improvementeven after 4 weeks of placebo therapy during the second course.

                  TABLE 8                                                         ______________________________________                                        CHANGES IN CLINICAL PARAMETERS IN PATIENTS                                    COMPLETING THE SECOND COURSE OF TREATMENT                                                Group A         Group B                                                       T.sub.2         Placebo                                                       (n = 7) *p      (n = 31)  *p                                       ______________________________________                                        Morning  Before  1.8 ± 0.2    2.5 ± 1.7                                 stiffness                                                                              After   0.8 ± 0.2                                                                            NS    1.3 ± 0.9                                                                          NS                                   (hours)                                                                       Joint    Before  7.9 ± 1.4    22.2 ± 2.4                                tenderness                                                                             After   11.0 ± 2.6                                                                           NS    13.5 ± 2.0                                                                         <0.001                               score                                                                         Number of                                                                              Before  4.2 ± 0.8                                                                            NS    7.0 ± 1.2                                 swollen  After   4.4 ± 0.9    3.5 ± 0.5                                                                          <0.05                                joints                                                                        Grip strength                                                                          Before  87.5 ± 8.0                                                                           <0.05 80.1 ± 9.2                                (mean of both                                                                          After   70.2 ± 9.5   97.1 ± 13.2                                                                        0.05                                 sides, mm Hg)                                                                 15 meter Before  20.3 ± 1.7                                                                           NS    31.5 ± 5.9                                walking time                                                                           After   17.1 ± 0.6   18.9 ± 2.3                                                                         NS                                   (second)                                                                      ______________________________________                                         *After vs before treatment                                               

Aside from grip strength, no significant changes were observed inclinical assessments in Group A patients after 4 weeks of placebotreatment.

As shown in Table 9, significant decreases in ESR and RF titer werenoted in Group B patients after the second course of treatment. Nosignificant worsening in laboratory parameters were noted in Group Apatients after 4 weeks of placebo therapy.

                  TABLE 9                                                         ______________________________________                                        CHANGES IN LABORATORY PARAMETERS IN PATIENTS                                  COMPLETING THE SECOND COURSE OF TREATMENT                                                 Group A        Group B                                                        T.sub.2        Placebo                                                        (n = 4)        (n = 25)  *p                                       ______________________________________                                        ESR (mm/hour)                                                                           Before  42.3 ± 6.0  68.5 ± 6.9                                          After   31.7 ± 7.3                                                                           NS   22.0 ± 4.9                                                                         <0.001                               RF (titers)                                                                             Before  49.3 ± 13.5 67.2 ± 12.1                                         After   32.0 ± 12.3                                                                          NS   32.0 ± 19.1                                                                        <0.05                                ______________________________________                                         *After vs before treatment                                               

The overall effectiveness of T₂ in the present trial was classified byits capacity to induce remissions, meaningful improvement or notherapeutic effect. (See Table 10).

                  TABLE 10                                                        ______________________________________                                        OVERALL EVALUATION OF THE PRESENT TRIAL                                                             Second                                                          First Course Treatment                                                                      Course Treatment                                                Group A Group B   Group A   Group B                                           T.sub.2 Placebo   Placebo   T.sub.2                                           (n = 27)                                                                              (n = 31)  (n = 24)  (n = 25)                                          No.  %      No.    %    No.  %    No.  %                              ______________________________________                                        Remission 2      7.4    0    0    0    0    0    0                            Improvement                                                                   Patient's 25     93     7    23   20   82   20   80                           assessment                                                                    Physician's                                                                             25     93     7    23   19   79   22   88                           assessment                                                                    Clinical  22     82     7    23   19   79   11   44                           criteria                                                                      Laboratory                                                                              23     85     4    13   18   75   13   52                           evaluation                                                                    ______________________________________                                    

Based on the therapeutic criteria for remission in RA developed by asubcommittee of the ARA, remission was observed in two patients of GroupA at the end of the first course of treatment.

The percentage of patients who experienced meaningful improvements wassignificantly higher for Group A than for Group B patients as evaluatedby physician's assessment, and clinical and laboratory evaluations afterthe first course of treatment.

The percent of Group B patients experiencing meaningful improvementafter the second course of treatment was also remarkable, whereasimprovement was maintained in Group A patients during the 4 week secondcourse of placebo.

In order to determine whether T₂ exerted an immunosuppressive effect inpatients with RA, peripheral blood mononuclear cells (PBMC) wereobtained from 18 patients of each group before and after the firstcourse of treatment. These cells were cultured for 14 days and theamounts of IgM-RF and total IgM secreted were determined using aradioimmunoassay. (See Table 11).

                  TABLE 11                                                        ______________________________________                                        PRODUCTION OF IgM-RF AND TOTAL IgM BY PBMC OF                                 PATIENTS AFTER THE FIRST COURSE OF TREATMENT                                             Group A  Group B                                                              T.sub.2  Placebo                                                              (n = 18) (n = 18)     *p                                           ______________________________________                                        RF      Before   7.2 ± 3.2                                                                             5.4 ± 1.6                                              After    1.5 ± 0.5                                                                             7.0 ± 2.2                                                                             <0.01                                  IgM     Before   220.7 ± 53.6                                                                          260.5 ± 49.3                                           After    151.9 ± 55.3                                                                          301.2 ± 100.5                                                                         <0.01                                  ______________________________________                                         *Group A vs Group B                                                      

In comparison with Group B, significant decreases in both IgM-RF andtotal IgM were noted in Group A after T₂ treatment. These resultssuggest that T₂ therapy had suppressed both IgM and IgM RF production inthese patients and thus exerted an immunosuppressive effect.

As shown in Table 12, the most common side effects of T₂ were dermalreactions including skin rash, cheilosis, thinning of skin and nails andpigmentation.

                  TABLE 12                                                        ______________________________________                                        INCIDENCE OF ADVERSE REACTIONS                                                        First Course Treatment                                                                     Second Course Treatment                                          Group A Group B  Group A   Group B                                            T.sub.2 Placebo  Placebo   T.sub.2                                            (n = 31)                                                                              (n = 31) (n = 24)  (n = 25)                                           No.  %      No.    %   No.  %    No.  %                               ______________________________________                                        Skin rash &                                                                             15     39     1    3   0    0    7    28                            cheilosis                                                                     Diarrhea  6      27     0    0   0    0    2    8                             Anorexia  2      5      0    0   1    4    0    0                             Abdominal pain                                                                          2      5      1    3   0    0    0    0                             Amenorrhea                                                                              5/16   31     0    0   5/16 31   1/18 6                             Postmenopausal                                                                          1/10   10     0    0   0    0    0    0                             vaginal                                                                       bleeding                                                                      ______________________________________                                    

Although the incidence of skin reactions was quite high in Group Aduring the first course of treatment, none of the patients had todiscontinue T₂ treatment. Amenorrhea was another important side effectof T₂. It was observed that 31% of female patients aged 49 or lesshaving received T₂ for 12 weeks developed amenorrhea whereas 6% ofpatients developed it after 4 weeks of T₂ treatment. Amenorrheadisappeared in most-patients when T₂ was discontinued.

FIG. 10 summarizes the assessed improvements in symptoms of rheumatoidarthritis described above. T₂ is an effective treatment for rheumatoidarthritis, significantly improving clinical manifestations andlaboratory correlates of inflammation. Although toxicity was frequent,it necessitated cessation of therapy in few. Clinical improvement wasobserved after only 4 weeks of therapy and persisted for at least 4weeks after the medication was discontinued. Therapy with T₂ suppressesthe in vitro production of IgM and IgM rheumatoid factor.

Administration of the T₂ extract has also been shown to be effective inthe treatment of systemic lupus erythematosus (Table 13). It alsoappears to be effective in relieving acute clinical manifestationsincluding joint inflammation, skin rash and renal disease (Table 13). Asteroid sparing effect of T₂ was also noted. In comparison withcorticosteroids and commonly used immunosuppressive agents, such ascyclophosphamide, patients treated with T₂ had fewer significantcomplications.

                  TABLE 13                                                        ______________________________________                                        THERAPEUTIC EFFECT OF T.sub.2 IN LUPUS NEPHRITIS                              ______________________________________                                        1.  Patient group                                                                 10 patients, aged 22-37, with duration of disease > 1 year                    were treated with T.sub.2                                                 2.  Laboratory evaluation - before treatment                                      +ANA: 10                                                                      anti-DNA binding >20%: 9                                                      Proteinuria >3 g/24 h: 10                                                     Elevated serum creatinine: 3                                              3.  Treatment plan:                                                               First month: T.sub.2 20 mg tid. Maintain prednisone <40 mg/day                Followed by T.sub.2 10 mg tid. and tapered prednisone                         Total course of T.sub.2 : 24 weeks                                        4.  Results of treatment:                                                         Serum creatinine returned to normal in 2/3                                    Proteinuria improved in 10/10:                                                undetectable: 3                                                               <1 g/24 h: 3                                                                  >1 g/24 h: 4                                                              Concomitant Medication                                                        3: withdrew frorn prednisone                                                  6: continued prednisone <10 mg/day                                            1: changed to cyclophosphamide                                                ______________________________________                                    

EXAMPLE 4 Components of T₂ Extract and Toxicity Thereof

The present example is provided to demonstrate the isolation andcharacterization of the various chemical components of a T. wilfordiiHook F root extract identified by the present inventors.

The structures of triptolide and tripdiolide are shown in FIG. 11. FIG.12 shows the structure of triptonide. Triptolide was isolated fromalcoholic extracts of Tripterygium wilfordii Hook F by the method ofKupchan et al.³¹. This scheme for triptolide preparation is outlined inFIG. 17. The present example demonstrates the effects of the T₂ extract(described in Example 1) or triptolide on the in cellulo of viability ofimportant immunopotent cells.

The effect of triptolide on immunopotent cells in vitro was determinedas follows:

T cells, B cells and fibroblasts (1×10⁶ /ml) were incubated with varyingconcentrations of T₂ or triptolide for 72 hr. The cells were assayed forcell viability by using a cytoflowmeter (FACSCAN) after the cells werestained with propidium iodine. Table 14 demonstrates the effect of T₂ ortriptolide on cell viability.

                  TABLE 14                                                        ______________________________________                                        EFFECT OF T.sub.2 OR TRIPTOLIDE ON CELL VIABILITY                             Inhibitors                                                                              T.sub.2 (μg/ml)                                                                          Tripolide(ng/ml)                                      Cell type                                                                             Control 0.1    1.0  10.0 100.0                                                                              0.1  1.0  10.0                          ______________________________________                                                  (Percent viable cells)                                              T cells 91.7    90.0   89.3 88.2 8.8  29.5 29.8 11.5                          B cells 55.6    50.9   44.3 30.5 10.6 20.9 20.9 15.6                          Fibroblasts                                                                           77.5    92.7   95.1 86.6 43.0 91.7 89.3 35.8                          ______________________________________                                    

T₂ at 100 μg/ml and triptolide at 10 ng/ml were toxic to fibroblastsindicating that at these levels, toxicity is nonspecific. At lowerlevels, suppression of T cell and B cell function is seen.

The capacity of triptolide to inhibit in vitro responses of humanlymphocytes was examined. As can be seen in table 15, triptolideinhibited proliferation of both T and B lymphocytes profoundly atconcentrations of 0.1-1.0 ng/ml.

                  TABLE 15                                                        ______________________________________                                                     PHA-Induced T Cell                                                                         SA-Induced B Cell                                   Concentration of                                                                           DNA Synthesis                                                                              DNA Synthesis                                       triptolide (ng/ml)                                                                         (.sup.3 H-Thymidine Incorporation, CPM)                          ______________________________________                                        0            93,400       7,900                                               0.1          24,200       2,000                                               1.0            100          100                                               ______________________________________                                    

Additional studies indicated that this triptolide fraction alsoinhibited the in vitro production of immunoglobulin from mitogenstimulated human B lymphocytes at comparably small concentrations. Theseresults demonstrate that the triptolide fraction is extremely toxic,however, its specificity of action is yet to be determined. Othercomponents of Tripterygium wilfordii Hook F include:

polpunonic acid (wilfortrine) (1) and the methyl ester thereof (2) shownin FIG. 13 and described by Keng et al. (Chem. Abst. 107: 55718y, p436,1987);

triptophenolide (1) and triptophenolide methyl ether (2) shown in FIG.14 and described by Wu et al. (Chem. Abst. 107: 96917f, p712, 1987);

triptonoterpenol shown in FIG. 15 and described by Deng et al. (Chem.Abst. 107: 112684k, p112692, 1987); and

wilformine (shown in FIG. 16), wilforine, wilforgine, and wilforzinedescribed by He et al. (Chem. Abst. 107: 130906p, 1987;

Purified components of the T. wilfordii extract that have essentiallyundetectable concentrations of triptolide will be administered topatients with autoimmune and inflammatory diseases including rheumatoidarthritis, systemic lupus erythematosus and psoriasis. Dosage will bedetermined based on the concentration of each therapeutic component inthe T. mixture.

EXAMPLE 5 Ethyl Acetate Preparation of Tripterygium Wilfordii Hook F

The present example is provided to demonstrate that the preparation ofTripterygium wilfordii Hook F may be obtained using a variety ofextraction protocols, including extraction by ethyl acetate.

An extract of the Tripterygium wilfordii Hook F root was preparedemploying an ethyl acetate extraction protocol. It is proposed that theethyl acetate extract will be administered orally in clinical use.

To prepare the ethyl acetate extract, roots of TWH obtained from FujianProvince of China were peeled and dried in the open air and in thesunlight. Roots obtained from other geographical areas are also expectedto be equally useful. The plant wood may also be dried using othertechniques including a low heat oven or incubator that will reachtemperatures of about 60° C. The woody parts of the root were ground toa powder. One thousand grams of the coarse powder of TWH were extractedwith 2500 ml of 95% ethanol for 24 hours. The extracted material wascollected in 5000 ml of 95% ethanol. The plant residue was refluxed with95% ethanol for 2 hours and the ethanol extract was combined with theinitial extract. The combination was evaporated under reduced pressureuntil all ethanol was removed. The concentrated ethanol extract wasdissolved in ethyl acetate with the aid of ultrasonification. The ethylacetate extract was filtered and the residue was dissolved with ethylacetate repeatedly. The ethyl acetate extract was combined, filtered andevaporated to dryness under reduced pressure. The material was groundinto a fine powder and mixed with starch. The mixed powder was furtherscreened through a #60 sieve.

The mixed powder will be incorporated into capsules suitable for humanuse using techniques well known to those of ordinary skill in the art⁶¹(see Remington's Pharmaceutical Sciences, 18th ed. (1990) for clinicaltrials which reference is specifically incorporated herein by referencefor this purpose). In some embodiments, one tablet will contain about 30mg of the ethyl acetate extract. The ethyl acetate extract containslittle triptolide, and the tablets will contain reduced amounts oftriptolide, defined for purposes of the present invention as preferablyno more than 10 μg to 20 μg of triptolide. Triptolide was measured withHPLC by comparison with a known standard of triptolide as described inExample 4 and FIG. 17. A single batch of 1400 gm was prepared andutilized for the pre-clinical evaluation described below.

Thin layer chromatographic scanner analysis carried out at SouthwesternMedical Center at Dallas showed that the average triptolide content ofthe Chinese EA extract from different batches manufactured by Huang ShiPharmaceutical Company was 1.33 μg per mg⁴⁴,45. The ethyl acetateextract produced by the present inventors contained much lowerconcentrations of triptolide of about 0.22 μg of triptolide per mg ofextract. Analysis by TLC and HPLC indicated that the Chinese and TexasEA extracts contained some similar components (FIG. 21).

Comparative Studies

Comparison of the diterpenes in the ethyl acetate extracts ofTripterygium wilfordii Hook F prepared in China and Texas was performed.330 mg of the Texas ethyl acetate extract and 40 mg of the Chinese ethylacetate extract were dissolved in ethyl acetate at a concentration of 66mg/ml and 8 mg/ml, respectively, followed by sonication for 25 minutesand filtration in vacuum. The ethyl acetate solution was passed througha 5 g neutral Al₂ O₃ column. The material was eluted with 30 ml ofethanol. After the ethanol elution was pooled with the ethyl acetatesolution, the mixed solution was evaporated under nitrogen air tilldryness. The residues were dissolved in 1 ml and 0.4 ml of chloroform,separately. 20 μl of each solution were applied to 20×20 cm silica gel GF 254 plate (polyester backing, 250 μm layer) and resolved withchloroform followed by chloroform/ether (1:4). Triptolide (3.6 μg),triptophenolide (10 μg) and tripdiolide (1.8 μg) served as referencestandards. After the plates were air-dried, the diterpenes werevisualized by the Kedde reaction. The relative concentrations oftriptophenolide, triptolide and tripdiolide in the Chinese and Texasethyl acetate extracts and a standard extract were performed. Theresults of this study are shown in FIG. 21.

*TEA: the ethyl acetate extract of Tripterygium wilfordii Hook Fprepared at UT Southwestern Medical Center at Dallas.

**CEA: the ethyl acetate extract of Tripterygium wilfordii Hook Fprepared in China.

As shown in FIG. 21, the Texas ethyl acetate extract evidenced a moreheavy staining band for triptophenolide than did the Chinese ethylacetate extract, yet about equal to that of the standard. In contrast,the band for triptolide was more intense for the Chinese extract ascompared to the Texas ethyl acetate extract. These findings demonstratethat there is more triptolide in the Chinese extract (because it is madefrom unskinned roots). Therefore the Chinese extract is more toxic.

Stability

The Texas ethyl acetate extract is stable at room temperature for atleast 1 year. Photosensitivity of the extract is unclear, and therefore,it is stored in the dark. It is expected that the preparation willremain stable for at least 8 years.

EXAMPLE 6 In Cellulo Activity of The Texas Ethyl Acetate Extract

Initial attempts to understand the mechanism of the action of the ethylacetate extract focused on its potential immunosuppressive activities.The present example demonstrates the significant in cellulo activity ofthe extract on antigen and mitogen-induced T cell proliferation and IL-2production.

The ethyl acetate extract was prepared as outlined in Example 5. Theethyl acetate extract exerted a number of immunosuppressive effects onhuman immune responsiveness, including antigen and mitogen-induced Tcell proliferation and IL-2 production. In cellulo (i.e., studies usingintact whole cells) studies were carried out to determine theconcentration of the extract that inhibited mitogen or antigen inducedhuman T cell proliferation and IL-2 production by T cells by 50% (ID₅₀).

Materials and Methods

Proliferation Studies: T cells were cultured with or without PHA (0.5μg/ml) in the presence or absence of the ethyl acetate extract at dosesof 5.7 μg/ml, 7.3 μg/ml, 0.08 μg/ml, 1.32 μg/ml, 0.7 μg/ml and 1.0 μg/mlfor 3 days. Significant effects on T cell proliferation were observed.

IL-2 Production Studies: The effects of the ethyl acetate extract onIL-2 production was examined. T cells were cultured with or without PHA(1 μg/ml) in the presence or absence of the extract at doses of 3.6μg/ml, 3.9 μg/ml and 0.83 μg/ml. Significant effects on IL-2 productionin the presence of either PHA or T.T. were observed (see Table 16).

The ethyl acetate extract at concentrations of 0.08-5.7 μg/ml inhibitedproliferation and IL-2 production by about 50%. The concentrations ofthe extract that induced death of about 50% of cells was also determined(LD₅₀). The LD₅₀ of the extract on the mitogen or antigen activated Tcells ranged from 17-70.5 μg/ml, which was 10-225 times thecorresponding ID₅₀ (Table 16). These results demonstrate that the TEAextract retained strong potency for immunosuppressive activities incellulo.

Much larger amounts of the ethyl acetate extract may be used with asignificantly reduced toxicity level. Therefore, the ethyl acetateextract would be expected to be relatively safer than the T₂ extract.

                  TABLE 16                                                        ______________________________________                                        IN CELLULO ID.sub.50 AND LD.sub.50 OF THE                                     ETHYL ACETATE EXTRACT ON HUMAN PBMCS                                                               Length of ID.sub.50                                                                           LD.sub.50                                Assay     Stimulus   Culture   (μg/ml)                                                                          (mg/per ml)                              ______________________________________                                        Proliferation                                                                           PHA (1 μg/ml)                                                                         2.5 days  5.7   70.5                                               PHA (1 μg/ml)                                                              +IL-2 (25 μ/ml)                                                                       2.5 days  7.3   74.2                                               T.T (10 μg/ml)                                                                        5 days    0.08  18.8                                               T.T (10 μg/ml)                                                             +IL-2 (25 u/ml)                                                                          5 days    1.32  17.0                                               SK (1 mg/ml)                                                                             5 days    0.7   20.0                                               SK +                                                                          (1 mg/ml)                                                                     +IL-2 (25 μ/ml)                                                                       5 days    1.0   19.8                                     IL-2 production                                                                         PHA (1 μg/ml)                                                                         24 hrs.   3.6                                                      PHA (1 μg/ml)                                                                         24 hrs.   3.9                                                      T.T. (10 μg/ml)                                                                       24 hrs.   0.83                                           ______________________________________                                         PHA, phytohemagglutinin; TT, tetanus toxoid; SK, streptokinase.          

EXAMPLE 7 In Vivo Animal Studies Of The Efficacy Of Ethyl AcetateExtract Of TWF

The present example describes studies that were conducted to demonstratethat the ethyl acetate extract of T. wilfordii exerts animmunosuppressive action on primary antibody responses in vivo.

In these studies, mice (C57 BL/6J) were immunized with TNP-BSAemulsified with complete Freund's adjuvant, according to techniques wellknown to those of skill in the art. The ethyl acetate extract wasprepared as outlined in Example 5. On the day of immunization, the micewere treated with the ethyl acetate extract at 125 or 250 mg/kg/dayorally. In other studies, mice were immunized with phosphorylcholine-KLHemulsified with complete Freund's adjuvant 30 days after the beginningof treatment with the ethyl acetate extract. Sera were harvested 10 and26 days after immunization. Antibodies against TNP, TNP-BSA or PC-KLH inthese sera were determined by the ELISA method. The ELISA method is astandard immunoreactivity assay well known to those of skill in the art.

Results from these studies show that the primary antibody responses toeach of these antigens were markedly decreased in the mice treated withthe ethyl acetate extract (Table 17). Treatment beginning 30 days beforeimmunization was the most effective at suppressing antibody responses,but treatment beginning on the day of immunization also significantly.diminished antibody responses.

                                      TABLE 17                                    __________________________________________________________________________    EFFECT OF TREATMENT OF MICE WITH THE TEA EXTRACT ON THEIR                     CAPACITY TO GENERATE PRIMARY ANTIBODY RESPONSES                                           Treatment with the Ethyl Acetate                                              Extract                                                                       Daily dose                                                                    (mg/kg of                                                                          before after  Antibody                                       Antigen     body immunization                                                                         immunization                                                                         Serum                                          Immunization                                                                         Assay                                                                              weight)                                                                            (days) (days) Dilution                                                                          OD                                         __________________________________________________________________________    PC-KLH PC-KLH                                                                             0    30     10     1:80                                                                              .250                                                   125  30     10         .133                                                   250  30     10         .084                                       TNP-BSA                                                                              TNP-BSA                                                                            0    0      10     1:320                                                                             .671                                                   125  0      10         .373                                                   250  0      10         .454                                                   0    0      26     1:5120                                                                            .528                                                   125  0      26         .364                                                   250  0      26         .282                                       TNP-KLH                                                                              TNP  0    0      10     1:20                                                                              .414                                                   125  0      10         .375                                                   250  0      10         .768                                                   0    0      26     1:320                                                                             .639                                                   125  0      26         .552                                                   250  0      26         .280                                       __________________________________________________________________________     C57 BL/6J mice (5 in each group) were treated without or with varying         doses of the ethyl acetate extract orally.100 mg of the antigens              emulsified in 0.1 ml of complete Freund's adjuvant were injected              intraperitoneally on the same day as the EA treatment started or 30 days      after the beginning of treatment with the TEA extract.Blood was taken fro     the tail vein on the 10th or 26th day after immunization.Antibodies           against TNP alone or TNPBSA or PCKLH in the sera were determined with         ELISA.Relative amount of antibodies in the sera was estimated by comparin     the O.D.readings of each sample at the same dilution for individual           assays.Multiple dilutions of serum were assayed and data shown for the        dilutions at which all readings were on the linear part of the curve.    

EXAMPLE 8 Toxicity of Ethyl Acetate Extracts

The present example is provided to demonstrate the reduced toxicity ofthe TWF preparations, particularly the ethyl acetate extract of thepresent invention as compared to other T. wilfordii extracts.

Acute toxicity testing was carried out using C57 BL/6J mice. For theinitial studies, 25 mice (5 in each group) were used to estimate theapproximate LD₅₀. No deaths developed with the ethyl acetate preparationof T. wilfordii until very high doses of 1200 mg/kg were administered.Eighty percent of the mice treated with 1400 mg/kg of the ethyl acetateextract died. Following this, 50 mice of the same strain were dividedinto 5 groups with equal numbers of each sex in each group. Mice weregiven a single dose of the ethyl acetate extract orally at 0, 1100,1150, 1230, 1350 and 1500 mg/kg body weight. The mice were observed for7 days thereafter. The LD₅₀ was estimated according to theSpearman-Karber Method⁶². LD₅₀ of the ethyl acetate extract from thisexperiment was 1253 mg/kg/day (Table 19). All death occurred within thefirst 3 days of the study.

                  TABLE 18                                                        ______________________________________                                        ACUTE TOXICITY TEST OF THE ETHYL ACETATE                                      EXTRACT IN MICE                                                               Dose (mg/kg)                                                                          Xi       Ri    Ni    Pi                                                                                 ##STR1##  I                                 ______________________________________                                        1100    3.04     0     10    0                                                                                 0.15       0.02                              1150    3.06     3     10    0.3                                                                               0.35       0.03                              1230    3.09     4     10    0.4                                                                               0.55       0.04                              1350    3.13     7     10    0.7                                                                               0.85       0.05                              1500    3.18     10    10    1.0                                              ______________________________________                                         50 mice (C57BL/6j) were randomly divided into 5 groups with equal numbers     of each sex for each group. The mice were treated with various doses of       the ethyl acetate extract as indicated orally for 7 days. The number of       dying mice was recorded. LD.sub.50 was calculated according to Spearman       Karber method:                                                                If X = LD.sub.50,                                                             Log X = X.sub.k - Σ (Pi + Pi + 1) × I × 0.5!.               Log X = 3.098                                                                 LD.sub.50 = 1253.1 mg/Kg.                                                     Xi = Log.sub.dose ; Ri = number of dying mice; Ni = number of tested mice     Pi = (Ri/Ni); I = Xi + 1 - Xi; X.sub.k = the logarithm of the dose (k) at     which at treated animals died.                                           

Autopsy was performed immediately after death of the mice. Histologicalexamination demonstrated marked lymphocytic necrosis of splenic germinalcenters and thymus, with only mild changes in liver, kidney, lung orbrain of some of the animals.

                  TABLE 19                                                        ______________________________________                                        COMPARISON OF THE TEXAS WITH THE CHINESE                                      ETHYL ACETATE EXTRACT                                                                       EA Extract                                                                    Texas     Chinese                                               ______________________________________                                        Source of plant material                                                                      Fujian province                                                                           Hubei province                                    Portion of the plant the                                                                      woody portion of                                                                          whole roots                                       EA extracted from                                                                             the roots                                                     Triptolide content                                                            (μg/gm of plant material)                                                                  4.80        27.50                                             (μg/mg of the EA extract)                                                                  0.22         1.33                                             ID.sub.50 (in vitro on PHA                                                                    5.7         2.0                                               stimulated human T cell                                                       proliferation, μg/ml)                                                      LD.sub.50 (on mice, mg/kg of                                                                  1253        764*                                              body weight)                                                                  ID.sub.50 in vitro T-cell                                                                     4.5 × 10.sup.-3                                                                     2.6 × 10.sup.-3                             proliferation/LD.sub.50                                                       ratio (on mice)                                                               ______________________________________                                         *The average of the LD.sub.50 of different batches of tablets of the CEA      extract prepared from TWH obtained from different counties or provinces o     China.                                                                   

As shown in Table 19, the LD₅₀ dose of the Chinese preparation is about764 mg/kg as compared to 1253 mg/kg for the Texas extract. The LD₅₀ ofT₂ in mice has been reported to be 159.7±14.3 mg/kg 40 and the LD₅₀ ofthe Chinese extract varied from 608-858 mg/kg. The ID₅₀ of the Chinesepreparation is 2.0 μg/ml as compared to 5.7 for the Texas extract. TheID₅₀ /LD₅₀ ratio of the Chinese extract is 2.6×10⁻³. This therapeuticactivity:toxic index value is significantly lower than the ID₅₀ /LD₅₀ratio of the TEA extract, ID₅₀ :LD₅₀ =4.5×10⁻³. The ratios of each ofthe extracts as calculated with the data presented in Table 19 indicatethat the Texas extract has a superior therapeutic activity:toxic indexbalance, and thus is superior as a therapeutic preparation compared topreparations of TwHF described in the literature.

EXAMPLE 9 In Vivo Use Of Texas Ethyl Acetate Extract

The present example is provided to outline the use of the Texas ethylacetate extract in animals, particularly humans.

The dosage schedule of the ethyl acetate extract to be used in initialescalation and safety studies will be calculated using the LD₅₀ of theextract and its triptolide content. The Texas ethyl acetate extract wasprocessed using the same procedure as used in China to produce theChinese ethyl acetate extract with the exception that the materialprepared as the Texas ethyl acetate extract is extracted from the peeledwoody portion of the roots of TWH. The Chinese extract is extracted fromthe whole root of the plant. The reported dosage of the Chinese extractwill be employed as a reference for calculating the dosage of the Texasethyl acetate extract (Table 19). The Chinese literature reports that60-120 mg/day of the CEA extract is safe and effective in the treatmentof RA (Shu et al., 1989; Hubei cooperative study group, 1981). Thisamount of the Chinese extract contains 131.2-262.4 μg of triptolide pertablet. Clinical trials with the Texas extract will employ escalatingdoses of 30 mg, 60 mg and 120 mg/day in three divided doses. The lowestdose is equivalent to approximately 25% of the smaller dosage of theChinese extract used safely in China whereas the highest dosage (120 mgdose of the TEA extract) approximates the lowest dosage of the CEAextract used in China and contains 26.4 μg of triptolide.

Administration of extracts of TWH are contraindicated in patients withleukopenia, thrombocytopenia, and impaired liver or kidney function.Studies have not been done to assess the effects of the extracts onpregnant or lactating women.

Patients will discontinue treatment with extracts of TWH if they developany of the following: persistent vomiting or diarrhea; profoundanorexia; WBC count of ≦2,500 cells/mm³, or platelet count of ≦100,000cells. Patients who develop these symptoms should be monitoredfrequently for white blood cell counts, as well as liver and kidneyfunction.

EXAMPLE 10 Fraction 924 of TWF-C Characterization and Identification

The present example is provided to detail the biological activity of afraction 924 component of a T. wilfordii ethyl acetate extract.

Based on previous examples, the present inventors were aware that manyditerpenoid compounds of TwHF, such as triptolide, possess suppressivecapacity on both in vitro and in vivo immune functions. The moleculargroups responsible for the immune suppressive function of thesecompounds are unknown, however, the core structure of diterpenoids whichmay be related to their activity consists of an α-β-unsaturated 5element lactone which can be identified by Kedde reagents. HPLC was usedto fractionate the ethyl acetate extract of TwHF and the Kedde reagentswere employed to trace the fractions. Then, the effect of selectedfractions on the in vitro IL-2 production and ³ H!-thymidineincorporation by PHA-activated T cells was determined. Fractions thatinhibit these T cell functions were selected and the chromatographicpattern was identified with HPLC. One fraction was further purified withHPLC and crystallized repeatedly; a pure, crystallized compound wasobtained and designated "924".

Purification of a 924 Fraction from an Ethyl Acetate Extract

The dried woody skinned part of the root of TwHF was extracted withethanol. The solution was concentrated at reduced pressure, and theresidue was dissolved in ethyl acetate. This solution was passed throughAl₂ O₃ with ethanol as an eluent. The eluate was evaporated and theresidue chromatographed on silica gel with chloroform, chloroform-etherand ethyl acetate as successive eluent. The fractions eluted withchloroform-ether were purified on a preparative HPLC column packed withNova-Pak C18 and equipped with a 214 detector. Methanol-water was themobile phase. A fraction having positive reaction to Kedde reagent wascollected and extracted with chloroform. The chloroform solution wasevaporated to dryness and the residue crystallized indichloromethane-hexane to yield 924. "924" is soluble in ethanol, andethanol and chloroform.

The "924" fraction inhibited DNA synthesis by T cells stimulated withPHA. The "924" fraction at a concentration of 1 ng/ml or more inhibitedPHA-stimulated T cells to uptake ³ H!-thymidine. The inhibition capacitywas correlated with the concentrations of the compound. The slope of theinhibition curve of "924" on T cell proliferation was quite flat in thatthe degree of inhibition changed from 14.6% to 54.2% when theconcentration of "924" increased from 1 ng/ml to 100 ng/ml. This wasdifferent from some components of TwHF, such as triptolide ortripdiolide which exerted potent immunosuppressive action with increasein its concentrations at the level of ngs, and resulted in significantenhancement of the inhibition of the cell function.

The "924" fraction inhibited IL-2 production by PHA-stimulated T cells.Similar with the pattern of the inhibitory effect of "924" on T cell DNAsynthesis, at the inhibitory concentrations, "924" was able to reducethe production of IL-2 by PHA-induced T cells. A 50% reduction of IL-2secretion was seen at 42.11 ng/ml (see FIG. 20B).

The 924 fraction also demonstrated significant activity in inhibitingproliferation of T cells, providing for inhibition of T cellproliferation at relatively low doses of 200 ng/ml of fraction 924 (seeFIG. 20C). At concentrations of 1,000 ng/ml fraction 924, no cellproliferation was evident (see FIG. 20C). There was no effect of "924"on IL-2R expression by PHA-activated T cells. Fraction "924" at 500ng/ml (at least 50 times the concentration effective to inhibit T cellproliferation or IL-2 production) did not affect the IL-2R expression byPHA-activated T cells.

Fraction "924" did not affect the viability of PHA-stimulated T cells atall employed concentrations, ranging from 1 ng/ml up to 1000 ng/ml. Thein vitro ID50/LD50 of "924" of T cell proliferation was more than 10indicating that, under the same cultural conditions, "924" did notincrease cell death until it reached to 10 times more than itsinhibitory concentration. The 924 fraction also demonstrated relativelylow cell toxicity, as demonstrated in FIG. 20D. Concentrations of the924 fraction of between 1 and 500 ng/ml did not differ significantly interms of cell viability, expressed as a percent of viable cells in thecontrol population. A 924 fraction concentration of 1,000 ng/ml was onlyslightly more toxic to cell viability as compared to the 0 ng/ml dose(see FIG. 20D, 0 ng/ml=22% of control; 1,000 ng/ml fraction 924=35% ofcontrol cell viability).

The positive reaction to Kedde reagent indicated that "924" has astructure of an α,β-unsaturated lactone. Since many of the knownditerpenoid compounds such as triptolide, triptochlorolide,16-hydroxytriptolide, tripdiolide, triptonide and triptophenolide alsohave the α,β-unsaturated lactone, the TLC and HPLC patterns of "924"were compared with these compounds. The pattern of "924" was differentfrom that of all of the above mentioned compounds. The pattern did nothave the chromatographic characteristics of diterpenoids and did notfollow the regular pattern of diterpenoids tested by several normal andreverse phase chromatographic systems. Therefore, "924" seems to be anon-diterpenoid compound. Upon further analysis of the 924 fraction byNMR/mass spectroscopy, the fraction was determined to be the purecompound, wilforonide (FIG. 18).

EXAMPLE 11 Immunosuppressive Effects of Wilforonide

T cells were cultured with or without PHA (0.5 micrograms/ml) in thepresence or absence of either, 10 ng/ml, 50 ng/ml, 100 ng/ml, 200 ng/mlor 500 ng/ml concentrations of the wilforonide preparation described inExample 10 for three days. ³ H!-thymidine was added for the last 14hours of culture. Each concentration was run in quadruplicate (4 times)and the results for each concentration averaged to provide a mean. Themean cpm×10³ values were combined and expressed as a percent of T cellinhibition. These data are provided at Table 20.

As shown in Table 20, a concentration of 100 ng/ml wilforonide provideda 21.19% inhibition of PHA-induced T cell proliferation in cellulo. Aconcentration of 200 ng/ml wilforonide provided a 26.43% inhibition ofPHA-induced T cell proliferation. A concentration of 500 ng/mlwilforonide resulted in a 62.86% inhibition of PHA-induced T cellproliferation (see Table 20). An ID₅₀ of 391.30 ng/ml wilforonide wasobserved (see Table 20).

                  TABLE 20                                                        ______________________________________                                        EFFECT OF WILFORONIDE ON PHA-INDUCED T CELL                                   PROLIFERATION*                                                                Wilforonide                                                                            cpm × 10.sup.3                                                                          Mean                                                 (ng/ml)  1.     2.     3.   4.   cpm × 10.sup.3                                                                  % inhibition                         ______________________________________                                        0        34.1   36.9   58.5 38.5 42.0                                         10       23.5   37.1   57.5 36.3 38.6    8.09                                 50       30.2   35.3   57.0 44.8 41.8    0.48                                 100      13.0   37.6   55.2 26.7 33.1    21.19                                200      6.8    33.9   56.5 26.4 30.9    26.43                                500      0.3    19.7   30.9 11.3 15.6    62.86                                ID.sub.50 (ng/ml)                        391.30                               ______________________________________                                         *T cells were cultured with or without PHA (0.5 μg/ml) in the presence     or absence of the indicated concentrations of wilforonide for 3 days.          .sup.3 H!-Thymidine was added for the last 14 hours. Data are from 4         independent experiments.                                                 

Effect of Wilforonide on PHA-Induced IL-2 Production

The effects of wilforonide on IL-2 production was also examined. T cellswere cultured with or without PHA (1 microgram/ml) in the presence orabsence of 0 (control), 10 ng/ml, 50 ng/ml, 100 ng/ml or 200 ng/mlwilforonide overnight. Cell-free supernatants were diluted 1 to 80 andthe IL-2 content assayed with CTLL-2 cells. IL-2 production by the Tcells cultured without stimulation was found to be less than 0.32units/ml.

The results obtained from the study are provided in Table 21. The 10ng/ml concentration of wilforonide evidenced a 37.94% inhibition ofPHA-induced IL-2 production relative to control. The 50 ng/mlconcentration of wilforonide resulted in a 54.22% inhibition ofPHA-induced IL-2 production relative to control. Concentrations of 100NG/ML resulted in a 68.20% inhibition, with the 200 ng/ml wilforonideconcentration resulting in a 74.96% inhibition of PHA-induced IL-2production.

An overall ID₅₀ of 42.11 ng/ml was also determined. These data areprovided in Table 21.

                  TABLE 21                                                        ______________________________________                                        EFFECT OF WILFORONIDE ON PHA-INDUCED                                          IL-2 PRODUCTION*                                                              Wilforonide                                                                           IL-2 (unit/ml)              %                                         (ng/ml) 1.      2.      3.    4.    Mean  inhibition                          ______________________________________                                        0       1.422   23.436  50.020                                                                              10.701                                                                              21.395                                    10      1.628   20.723  27.374                                                                              3.385 13.278                                                                              37.94                               50      1.664   26.983  4.509 6.042 9.795 54.22                               100     0.800   11.160  10.170                                                                              4.982 6.803 68.20                               200     0.850   10.301  8.502 1.773 5.357 74.96                               ID.sub.50 (ng/ml)                         42.11                               ______________________________________                                         *T cells were cultured with or without PHA (1 μg/ml) in the presence o     absence of the indicated concentrations of wilforonide overnight. Cellfre     supernatants were diluted 1 to 80 for IL2 content assay with CTLL2 cells.     IL2 production by the T cells cultured without stimulation was less than      0.32 unit/ml.                                                            

Inhibitory Effect of Wilforonide on Antigen-Induced T Cell Proliferation

T cells were cultured with or without SK (1 ng/ml) or SK+IL-2 (50 u/ml)or SK+PMA (0.2 ng/ml) in the presence or absence of the followingconcentrations of wilforonide: 10 ng/ml, 50 ng/ml, 100 ng/ml, 500 ng/mlor 1,000 ng/ml. The cells were allowed to culture for five days. Thecultures were pulsed with ³ H!-thymidine for the last 24 hours ofculture. The data collected from this study is provided in Table 22.These data represent the mean of the percent inhibition of ³H!-thymidine incorporation of five independent experiments. ID₅₀ werecalculated based on the regression formula by using the fx-3600calculator. T cells cultured with SK or SK+IL-2 or SK+PMA gave cpm of1.87×10³, 4.99×10³ and 6.67×10³, respectively.

The response to SK inhibited by low concentrations Wilforonide is shownto be partially overcome by adding IL2 or PMA, as demonstrated by themarkedly higher (increased) ID₅₀. This indicates that inhibitioncorrelates to a decrease in IL2 production. This decrease is overcome byadding IL2 or co-stimulating with PMA that induces IL-2 production.

The data from this study is provided in Table 22.

                  TABLE 22                                                        ______________________________________                                        INHIBITORY EFFECT OF WILFORONIDE                                              ON ANTIGEN-INDUCED T CELL PROLIFERATION*                                      Wilforonide                                                                   (ng/ml)   SK           SK + IL-2                                                                              SK + PMA                                      ______________________________________                                        10        42.15        19.59    22.67                                         50        44.47        12.37    17.70                                         100       49.14        14.52    20.32                                         500       75.20        61.70    53.30                                         1,000     84.23        86.21    65.43                                         ID.sub.50 (ng/ml)                                                                       127.12       476.00   613.00                                        ______________________________________                                         *T cells were cultured with or without SK(1 mg/ml) or SK plus IL2(50          μ/ml) or SK plus PMA(.2 ng/ml) in the presence or absence of indicated     concentrations of wilforonide for 5 days. Cultures were pulsed with            .sup.3 H!-thymidine for the last 24 hours. Data represent the mean of th     % inhibition of  .sup.3 H!-thymidine incorporation of 5 independent           experiments. ID.sub.50 were calculated based on the regression formula by     using the fx3600 calculator. T cells cultured with SK or SL + IL2 or SK +     PMA gave cpm of 1.87 × 10.sup.3, 4.99 × 10.sup.3 and 6.67         × 10.sup.3, respectively.                                          

EXAMPLE 12 TwHF T2 Extract Inhibits Production Of Interferon γ andTranscription Of The IL-2 Gene

The present example demonstrates that the T2 extract of Tripterygiumwilfordii Hook F inhibits production of interferon γ and transcriptionof the IL-2 gene.

Transcription studies were carried out to determine whether the extractof TwHF directly affected IL-2 gene transcription or hadpost-transcriptional effects. Jurkat cells that had been stablytransfected with a construct containing the IL-2 promoter drivingtranscription of the reporter gene, chloramphenicol acetyltransferase(CAT), were employed.

Methods

IFN-γ assay. IFN-γ content in supernatants was determined with aradioimmunoassay kit as described by the manufacturer (Centocor,Malvern, Pa.). T cells (1×10⁵ /ml) were incubated with PHA (1 μg/ml) for24 h in the presence or absence of the EA extract. Cell-freesupernatants were assayed for IL-2 and IFN-γ content.

IL-2 CAT assay. Jurkat cells were electroporated with a construct of theIL-2 promoter containing most of the T cell specific transcriptionfactor binding sites within the promoter/enhancer (region -342 to +47derived from IL-2/pJGFCA19 construct) driving the bacterialchloramphenicol acetyl transferase (CAT) gene inserted into a vectorcontaining a neomycin selection marker, designated IL-2/PML3 (72,73).The cells were selected with geniticin (0.5 mg/ml) for approximately onemonth before use. The cells were then incubated with PEA and PMA, in thepresence of varying concentrations of an extract of TwHF (achloroform/methanol extract from the woody portion of the roots of TwHF,dissolved in DMSO and diluted with culture medium) or in medium alonefor 20 h. After incubation, the supernatants were collected for assay ofsecreted IL-2 and equivalent numbers of cells were washed and lysed byrepeated freeze/thaw in 100 μl of 0.25M Tris-HCl (pH 7.8) andcentrifuged for 5 minutes at high speed to remove cellular debris. CATactivity was measured by the addition of 1 μCi ¹⁴ C!-chloramphenicol (60mCi/mmol; New England Nuclear, Boston, Mass.) and 14 μl of 5 mM acetylcoenzyme A to the lysates followed by a 12 h incubation at 37° C. aspreviously described by Gorman et al. (74). The reaction was stopped bythe addition of 1 ml cold ethyl acetate and after phase separation, theorganic phase was evaporated, resuspended in 20 μl of ethyl acetate andspotted onto silica gel thin layer chromatography plates. Thechromatograms were visualized and the radioactivity quantified using anautomated β-detection and imaging device (AMBIS). The percent conversionwas calculated by dividing the cpm of the acetylated chloramphenicol bythe total chloramphenicol present in each sample. CAT activity was foundto correlate with levels of secreted IL-2 when these cells werestimulated with various mitogens. Jurkat cells transfected withPML-LDLr-6500 were taken as a positive control for CAT activity⁷⁵.

Results

Inhibition of mitogen-induced IL-2 gene transcription by extracts ofTwHF. The data of Table 23 show that the extract of TwHF inhibitedmitogen-induced CAT activity in a dose-dependent manner, similar to thatobserved for IL-2 production.

                  TABLE 23                                                        ______________________________________                                        Inhibition of IL-2 Transcription by a T2 Extract.sup.1                                               IL-2 Production                                                      % Acetylation                                                                          (Units/ml)                                             ______________________________________                                        Control         0.17       23.4                                               PHA + PMA       6.90       5440                                               pHA + PMA + T2 1 μg                                                                        0.22       0                                                  pHA + PMA + T2 5 μg                                                                        0.22       0                                                  pHA + PMA + T2 10 μg                                                                       0.19       0                                                  4JK cells       95.80      --                                                 ______________________________________                                         .sup.1 Jurkat T cells stably transfected with an IL2 promoter/CAT             construct, were incubated with or without PHA (2 μg/ml) in the presenc     or absence of varying concentrations of an extract of TwHF for 18 h.          Supernatants were collected for IL2 assay with CTLL2 cells. Cellular          extracts were assessed for CAT activity as described in the methods. CAT      activity of a control cell line transfected with a CAT construct driven b     a constitutively active promoter (4JK cells) is shown as a positive           control for the assay.                                                   

These results indicate that the extract of TwHF inhibits transcriptionof the IL-2 gene.

Inhibition of IFN-γ production. As shown in FIG. 34, the EA extractinhibited PHA-induced IL-2 production (EC₅₀ =0.75±0.12 μg/ml).PHA-induced IFN-γ production was also very sensitive to EA-mediatedinhibition (EC₅₀ =0.64±0.04 μg/ml).

EXAMPLE 13 Glucocorticoid Receptor Binding Of Components Of TripterygiumWilfordii Hook F (TwHF)

The present example provides data on the capacity of the TwHF extractand purified TwHF components to bind to the glucocorticoid receptor(GR). Binding of the TwHF extract and components thereof to the GR wasdemonstrated by competitive inhibition of binding of the natural GRligand using several model systems. The ability of TwHF to compete with³ H! dexamethasone for binding to the glucocorticoid receptor, theability of TwHF to inhibit glucocorticoid receptor-mediated activationof a responsive target gene construct in transfected mammalian cells,and the capacity of TwHF to inhibit growth of a transfected cell lineexpressing a glucocorticoid receptor regulating the SV40 large Tantigen, and therefore, whose growth is dependent on dexamethasone, wereexamined.

Chloroform-methanol extracts of TwHF were used in this example; theactivity of these extracts is correlated to the activity of the ethylacetate extracts by the amount of triptolide in the preparations. Theamount of triptolide was measured by HPLC as described in Example 15.

Methods

Whole cell glucocorticoid binding assay. Human skin fibroblasts werecultured in MEM-10% bovine calf serum in an atmosphere of 5% CO₂ at 37°C. Cells were plated in 7 cm wells at 750,000 cells per well.Twenty-four hours before experiments, the medium was changed to MEM with5 mg/ml bovine serum albumin. On the day of the experiment, the mediumwas replaced with 3.0 ml of MEM per well with 10 nM 3h! dexamethasone.Varying amounts of the extract of TwHF were added to some wells . Thecells were incubated at 37° C. for 60 minutes, then washed, harvested bytrypsinization, and lysed in 1 ml water. Aliquots were assayed forprotein and counted for bound radioactivity. These cells express, onaverage, about 170 fmol of glucocorticoid receptor per mg protein.

Cell transfections and assays of GR mediated gene activation. COS-7cells were maintained in culture in DMEM-10% bovine calf serum. Thesecells do not express endogenous steroid hormone receptors. Cells wereplated at 10⁶ cells per 10 cm plate. Twenty-four hours later, the cellswere transfected with 500 ng pRShGrα, a GR expression vector (obtainedfrom the Salk Institute, La Jolla, Calif.) and 10 μg ofpMMTV-luciferase, a synthetic reporter gene. However, other expressionvectors and synthetic reporter genes available to those of skill in theart may be used for transfection. The vector pMMTV-luciferase containsthe luciferase gene under regulatory control of glucocorticoid-inducibleelements in the MMTV-long terminal repeat. Cells were transfected usingthe calcium-phosphate precipitation technique (mammalian transfectionkit, Stratagene, La Jolla, Calif.). After transfection, dexamethasone,(1 μM) was added to the medium. Twenty-four hours later, the cells werewashed, harvested, and lysates prepared by three cycles offreeze/thawing. The lysates were cleared by centrifugation at 12,000×gfor 5 minutes; aliquots were assayed for protein content and equalamounts of proteins were used for luciferase assays.

An additional assay system that was employed involved IDH4 cells, whosegrowth is dependent on the presence of dexamethasone. IDH4 cells, thatexpress endogenous glucocorticoid receptors, were produced bytransfecting human fibroblasts with a plasmid containing the MMTV-longterminal repeat driving the SV40 large T antigen. Since extended growthof these cells is dependent on expression of the large T antigen, thesecells require exogenous glucocorticoid to maintain proliferation.

Results

TwHF extract binds GR. The chloroform-methanol extract of TwHF inhibitedbinding of ³ H! dexamethasone to the GR endogenously expressed in humanskin fibroblasts (FIG. 22). Intact monolayers of human genital skinfibroblasts (about 170 fmol GR per mg protein) were incubated with 10 nM³ H! dexamethasone alone or with an increasing concentration of TwHF. Noestimate of the relative binding affinities of TwHF and dexamethasone ispossible, however, since the TwHF extract is a mixture of compounds.Inhibition of 50% of GR activity occurred with the addition ofapproximately 5 μg of the extract. Addition of pure ethanol, the solventfor the TwHF extract, in equal volumes had no effect on GR ligandbinding activity. Moreover, the TwHF extract had no effects on bindingof dihydrotestosterone to genital skin fibroblasts, indicating that itdid not inhibit androgen receptor activity, implying a specific effecton GR.

TwHF extract inhibits dexamethasone-induced activation of target genetranscription but lacks intrinsic GR agonist activity. Thechloroform-methanol extract of TwHF also inhibited GR-mediatedactivation of a synthetic target gene (FIG. 23). COS-7 cells weretransfected with a human GR expression vector and the MMTV-luciferasereporter gene construct. A parallel set of plates was transfected withthe constitutively active RSV-luciferase construct. After 24 hours,dexamethasone was added at a concentration of 10 nM alone or incombination with increasing concentrations of TwHF. Cells were lysedafter 24 hours and assayed for luciferase activity. In the absence ofTwHF, background levels of luciferase activity were about 1% ofdexamethasone-induced activity. Addition of dexamethasone resulted in,on average, a 100- to 200-fold induction of luciferase activity.Addition of the TwHF extract in concentrations shown to compete for GRbinding inhibited target gene activation (FIG. 23). Luciferaseexpression under control of a constitutively active promoter, i.e., apromoter not responsive to glucocorticoid receptor, (RSV-luciferase) wasnot inhibited by the addition of TwHF (FIG. 23) indicating specificityof TwHF extract for GR dependent processes.

To determine whether TwHF had direct glucocorticoid agonist activity,COS-7 cells transfected with a human GR expression vector and the MMTVreporter construct were stimulated with TwHF, but without dexamethasone.TWF caused no direct target gene activation (Table 24). By contrast,dexamethasone induced dramatic reporter gene transcription.

                  TABLE 24                                                        ______________________________________                                        TWF Lacks Intrinsic GR Agonist Activity                                                TWF      TWF      TWF    TWF    DEX                                  Control  0.4 μg/ml                                                                           0.8 μg/ml                                                                           2.0 μg/ml                                                                         4.0 μg/ml                                                                         10 nM                                ______________________________________                                        RLU   998    1260     1603   1167   903    193,419                            #1                                                                            RLU  28S0    2012     1905   1106   517    218,786                            #2                                                                            ______________________________________                                         COS-7 cells were transfected with human GR expression vector and the          MMTVluciferase reporter gene construction. After 24 hours TWF at              concentrations known to compete for GR binding (0.4-4.0 μg/ml) were        added. Cells were lysed after 24 hours and extracts assayed for luciferas     activity. Controls included no addition (negative control) and 10 nM          dexamethasone (positive control). Data shown are relative light units         (luciferase activity) for duplicate transfections in a single experiment.

Similar experiments in L929 cells, and in both COS and L929 cellsstimulated with cAMP (known to "unmask" agonist activity of some GRcompetitors) confirmed this lack of agonist function of TwHF.

To compare the concentration response curves for TwHF-mediatedinhibition of GR ligand binding activity and TwHF inhibition ofGR-mediated target gene activation, the data of FIGS. 22 and 23 werecombined. Whereas the curves are comparable, TwHF appears several-foldmore potent for inhibition of target gene activation compared to ligandbinding inhibition (FIG. 24).

The extract of TwHF and the purified component tripdiolide inhibiteddexamethasone dependent growth of IDH4 cells in a concentrationdependent manner (FIGS. 25A, 25B and 25C). Tryptophenolide was lessactive and its activity was not generally correlated with dosage. IDH4cells (1×10⁶ /ml) were cultured for 72 hours in DMEM supplemented with10% of dexamethasone-free bovine serum with or without dexamethasone inthe presence or absence of T2 or tripdiolide or triptophenolide, asindicated. ³ H!-Thymidine was present for the last 16 h. Data representthe mean k inhibition of ³ H!-thymidine incorporation of three replicatedeterminations of three separate experiments. IDH4 cells incubatedwithout stimulation gave 16,391 cpm. Cells cultured with dexamethasoneat 1 nM, 10 nM, 100 nM and 1,000 nM gave 35,554, 52,937, 105,357 and96,677 cpm, respectively.

Further studies assessed whether cortisol-like immunoreactivity wascontained in TwHF extract. With most anti-cortisol antibodies tested, noevidence for the presence of such molecules in TwHF was found. This wasconfirmed by fractionation experiments on Sephadex LH-20 in which theglucocorticoid receptor blocking capacity of TwHF was found to exist infractions distinct from the elution position of authentic cortisol. Witha single anti-cortisol antibody directed against the A ring of thesteroid, some cortisol-like immunoreactivity was detected (FIG. 26).These data suggest that some components of TwHF could have cortisol-likestructural features, but no authentic cortisol was found.

Purified TwHF compounds were similarly tested for effects on ligandbinding to GR, and for inhibition of GR-mediated target gene activation.Both triptolide (compound A) and tripdiolide (compound B) inhibitedtarget gene activation by GR, whereas triptophenolide (compound C) wasfar less potent. Of importance, triptophenolide is much lessimmunosuppressive than triptolide or tripdiolide. Binding inhibition andtarget gene activation curves are shown for compound A (FIG. 27). Again,the compound was more potent at inhibition of MMTV-luciferase inductionby more than an order of magnitude. None of the purified TwHF componentsshowed cortisol immunoreactivity (FIG. 28.)

EXAMPLE 14 Anti-Inflammatory Properties Of TwHF Extract

In the present example, the extract of TwHF and tripdiolide wereexamined for their ability to inhibit the in vivo induction of apro-inflammatory enzyme activity, namely the stimulation of theinducible form of cyclooxygenase (COX-2). Cyclooxygenase-1 is the enzymeresponsible for the constitutive synthesis of prostaglandins and certainrelated autocoids, mediators of inflammation. Inhibition of this enzymecauses the side effects of NSAIDS. During inflammation and afterstimulation, inflammatory cells, such as macrophages, produce a newenzyme, cyclooxygenase-2, that is responsible for much of theprostaglandin production at inflammatory sites.

Monocytes (1×10⁶ /ml), separated from normal human PBMCs, were incubatedin RPMI-1640 medium supplemented with 5% normal human serum with orwithout LPS (10 μg/ml) and in the presence or absence of one of thefollowing reagents: T2, tripdiolide, dexamethasone, or RU486, at theindicated concentrations (FIGS. 29A-29D). After 18 hours of incubation,cell-free supernatants were collected and assayed for PGE2 content witha radioimmunoassay kit (Amersham), as described by the manufacturer.Data represent the mean of two replicate determinations of two separateexperiments. Monocytes cultured without LPS produced 392 pg PGE2/0.2million cells. Human monocytes produce prostaglandinE₂ (PGE₂) whenstimulated with bacterial endotoxin. This relates to the induction ofcyclooxygenase 2 (COX-2) and is inhibited by dexamethasone (FIGS.29A-29D). Similarly, the production of PGE₂ is inhibited by the extractof TwHF and its purified immunosuppressive component, tripdiolide.Constitutive PGE₂ production by unstimulated monocytes that is mediatedby cyclooxygenase-1 was not inhibited by TwHF or its components.Specificity is shown in that previous studies had documented that theextract of TwHF did not inhibit other functions of monocytes, such asantigen presentation.

In summary, the data are consistent with the conclusion that componentsof TwHF specifically interact with the glucocorticoid receptor and exertanti-inflammatory and immunosuppressive effects by this mechanism, andhave no direct agonist activity on glucocorticoid responsive genes.

These data further demonstrate that the T2 extract, and componentsthereof, bind the glucocorticoid receptor, and the bound complex failsto activate glucocorticoid receptor-sensitive promoter regions. Genesthat require those promoters to be active, therefore, are not induced.Concomitantly, T2 inhibits inflammatory processes as demonstrated hereinby the inhibition of induction of cyclooxygenase-2 and also by theinhibition of IL-2 and interferon γ gene activation. The combined effectof anti-inflammatory property without induction of steroidal-relatedagonist activity provides a heretofore unknown and long-sought treatmentmethod for inflammation, autoimmune disease and other immunosuppressiveconditions where the undesirable effects of steroids (TwHF has noagonist activity) and non-steroidal anti-inflammatory drugs (e.g.,aspirin) can be avoided (cyclooxygenase-1 is not inhibited by TwHF).

EXAMPLE 15 HPLC Determination Of Triptolide And Tripdioloide In An EthylAcetate Extract Of TwHF

A new analytical method for the determination of triptolide andtripdiolide in ethyl acetate extracts of Tripterygium wilfordii Hook F.is described in the present example. The procedure consists ofpreliminary enrichment of the triptolide and tripdiolide by Sep-Pakalumina B cartridge chromatography followed by HPLC analysis. HPLC isperformed with a stainless steel column packed with Nova-Pack C18, usingacetonitrile-water (19:81) as a mobile phase for triptolide andacetonitrile-water (11:89) for tripdiolide. The effluent is monitored byultraviolet detection at 214 nm. Quantitative analysis of triptolide isthen carried out by comparison to an internal standard, and oftripdiolide by the external standard method. The amounts of triptolideand tripdiolide per 100 mg of the ethyl acetate extract were determinedto be 19.88 μg and 9.58 μg respectively. The method is sufficientlysensitive and specific to assay the diterpenes found in Tripterygiumwilfordii Hook F. accurately.

Methods

Instruments. The Waters (Milford, Mass.) liquid chromatograph employedwas configured with two Model 510 pumps, a Model U6K injector and Model441 UV detector set up at 214 nm. The data was processed with Millenniumsoftware, Version 1.10 (Waters Assoc.). The stainless steel column (150mm×3.9 mm I.D.) was packed with Nova-Pak C18 particle size 4 μm (WaterAssoc.). An HPLC pre-column, with an insert packed with Nova-Pak C18,(Water Assoc.) was used to extend the column life. The model ULTRAsonik2QT/H ultrasonic water bath used in the solvent degassing and samplepreparation was purchased from NEY Barkmeyer Division (Yucaipa, Calif.).

Chemicals and reagents. Triptolide and tripdiolide were prepared fromthe ethyl acetate extract of TwHF by silica gel column chromatographysuccessively with chloroform, chloroform-ether and chloroform-ethylacetate as the eluents. The fractions containing triptolide andtripdiolide were purified on preparative HPLC with a Nova-Pack C18column, 25×100 mm, using acetonitrile-water as the mobile phase. Thecompounds were recrystallized from n-hexane-dichloromethane. Triptolidewas identified by V, IR, proton NMR and mass spectrums. Tripdiolide wasidentified by HPLC, TLC and proton NMR and comparison with the knownlaboratory product provided by Boehringer Ingelheim Pharmaceuticals Inc.(Ridgefield, Connecticut). Acetonitrile was HPLC grade purchased fromAldrich Chemical Co. (Milwaukee, Wis.), water was Millipore pure, andother solvents were GR grade. The mobile phases were degassed by vacuumin conjunction with sonication just before use. The Sep-Pak Plus aluminaB cartridge was purchased from Waters Assoc. (Milford, Mass.);acetophenone, selected as an internal standard for the triptolideassessment, was purchased from Sigma Chemical Co. (St. Louis, Mich.).The chemical structures of triptolide and tripdiolide are shown in FIG.11.

Preparation of the ethyl acetate extract of TwHF. The roots of TwHF werecollected from Fujian province, China. The skin was removed from theroots and the woody portion of the roots was ground to coarse powder.1000 g of the coarse powder was extracted with ethanol three times. Theethanol solutions were combined and evaporated under reduced pressure.The residue was then extracted with ethyl acetate. Concentration of thesolution under reduced pressure yielded 22 g of the ethyl acetateextract.

Enrichment procedure. About 50 mg of the ethyl acetate extract wasweighed accurately and dissolved in 10 ml of chloroform in an ultrasonicbath for 25 minutes. The extract solution was filtered and the residuewas washed with 10 ml of chloroform-ethyl acetate (9:1). The washingscombined with the original chloroform solution were applied to theSep-Pak cartridge. 25 ml of chloroform-ethyl acetate (9:1) and 15 ml ofethyl acetate-methanol (9:1) were successively passed through thecartridge. The chloroform-ethyl acetate fraction, used for thedetermination of triptolide, was evaporated to dryness under a gentlestream of nitrogen. The residue was dissolved with 1.00 ml ofacetophenone solution, that was prepared by dissolving acetophenone inmethanol to obtain a solution having a concentration of 12.5 μg per ml.The dissolved residue was diluted with acetonitrile-water (19:81) to2.00 ml. The ethyl acetate-methanol fraction was evaporated. The residuewas dissolved in 1.00 ml of acetonitrile-water (11:89) solution and usedto analyze for tripdiolide content.

Determination of diterpenes. A 10 μl volume of each purified samplesolution was injected into the liquid chromatograph. The mobile phasefor each separation is listed with the individual chromatogram.Triptolide was determined by comparison to an internal standard. Thereference solutions containing 1.83, 3.66, 7.32, 16.08 and 36.18 ng μl⁻¹of triptolide and 6.25 ng μl⁻¹ of acetophenone for each solution wereprepared in acetonitrile-water (19:81). The reference solutions oftripdiolide were prepared in acetonitrile-water (11:89) at theconcentrations of 1.28, 2.55, 5.10, 10.20, 20.40, 30.60, and 40.80 ngμl⁻¹. Two replicates of each were injected into the HPLC system. Theresulting chromatograms yielded data for the standard curves. Thecontents of triptolide and tripdiolide were calculated and expressed per100 mg of the dried extract (drying at 80° C. to a constant weight.

Results

The enrichment procedure and chromatographic separation as well as theselection of an internal standard are three major problems in HPLCanalysis of crude plant extracts. Many enrichment procedures wereinvestigated during the preliminary phases of this study. These includeddifferent absorbents, such as silica gel, alumina N, florisil, diol,aminopropyl NH₂, cyanopropyl CN, activated carbon and polyamide. Inaddition, different solvent systems were tested. Sep-Pack Plus alumina Bcartridge was found to be an efficient and convenient purifying methodthat involved the minimum number of steps. HPLC was performed with aNova-Pack C18 column using acetonitrile-water as a mobile phase system.This resulted in a better separation of triptolide, tripdiolide andacetophenone from other components of the plant than did the use ofmethanol-water as a mobile phase. A detective wavelength of 214 nm wasemployed because of the α, β-unsaturated lactone ring in the diterpenestructures. Acetophenone was found to be the most suitable internalstandard for the determination of triptolide. Because of interferencefrom other components, attempts to use an internal standard in thedetermination of tripdiolide were unsuccessful. FIG. 30 illustrates thechromatogram of triptolide and acetophenone. The retention times of thetwo compounds were 11.35 min. and 8.15 min. respectively. FIG. 31 showsthe chromatogram of tripdiolide. The retention time was 10.3 min.

The separation of triptolide, acetophenone and tripdiolide from theextracts of TwHF by HPLC was achieved using the method described above.This approach provided a good quantitative and reproducible recovery.FIG. 32 depicts a typical chromatogram of the extract for thedetermination of triptolide after addition of acetophenone. It isapparent that the other components present in the extract did not alterthe internal standard peak. FIG. 33 shows a typical chromatogram of theextract for the determination of tripdiolide.

The peak purity was tested by collecting the fractions corresponding toboth of the compounds and analyzing them by HPLC on the same columnusing methanol-water (30:70) as a mobile phase and adjusting the flowrate to 1.0 mL per min. The results indicated that a single componentwith the retention times corresponding to triptolide (5.1 min.) ortripdiolide (16.7 min.) had been isolated.

A linear calibration graph for triptolide was obtained by plotting theratio of the peak area of triptolide to the internal standard (y) versusthe amount of triptolide (x, ng). The regression equation andcorrelation coefficient (r) were y=0.025x -0.049, r=0.99999, n=5. Thelinear calibration graph of tripdiolide was obtained by plotting thepeak area response of tripdiolide (y) versus the amount of tripdiolide(x, ng). The regression equation and correlation coefficient werey=744.2x -2123, r=0.9998, n=7. The range of the calibration curve wasfrom about 18.3 ng to about 361.8 ng for triptolide and from about 12.8ng to about 408.0 ng for tripdiolide.

The detection limit was determined at very low concentrations using thedescribed method. The detectable amounts of triptolide and tripdiolidewere about 4.77±0.66 ng (n=4) and about 9.05±0.66 ng (n=3) respectively.

The recovery test was carried out by adding pure triptolide andtripdiolide to the extract and assaying with the same proceduredescribed above. The recoveries (mean %±SD) of triptolide was about98.34±1.54 (n=4) and tripdiolide was about 95.85±1.49 (n=4).

The assay results are displayed in Table 25. Each term is the mean oftwo injections. The contents of triptolide and tripdiolide in about 100mg of the ethyl acetate extract of TwHF were about 19.88 and about 9.58μg respectively.

                  TABLE 25                                                        ______________________________________                                        CONTENTS OF TRIPTOLIDE AND TRIPDIOLIDE IN THE                                 EXTRACT OF TRIPTERYGIUM WILFORDII HOOK F.                                     DETERMINED BY HPLC.sup.1                                                              Amount in individual     Relative                                             Determinations (μg per                                                                              Standard                                     Diterpenes                                                                            100 mg of extract)                                                                           Mean ± SD                                                                            Deviation (%)                                ______________________________________                                        Triptolide                                                                            18.45 20.90 21.05 18.95                                                                      19.88 ± 1.04                                                                         0.052                                                19.82 20.12    n = 6                                                  Tripdiolide                                                                           10.30 10.03 10.17 9.64                                                                       9.58 ± 0.54                                                                          0.056                                                9.72 10.14 10.14 9.51                                                                        n = 15                                                         9.12 8.75 8.98 9.01                                                           8.98 9.11 10.06                                                       ______________________________________                                         .sup.1 The values from individual experiments are the amounts per 100 mg      of dry ethyl acetate extract.                                            

In summary, this example provides an accurate, sensitive and reliablemethod for the determination of triptolide and tripdiolide in an extractof TwHF. The pretreatment of samples with the Sep-Pak alumina Bcartridge before HPLC represents a fast, simple and effective enrichmentprocedure with a very satisfactory recovery of the compounds. Thesuccessful employment of the internal standard greatly improved theaccuracy and reproducibility for the triptolide assay. Triptolide andtripdiolide are two of the major diterpene compounds contained in TwHF.This study provides the first quantitative data about the tripdiolidecontent in TwHF. Combined with the capacity to analyze triptolide, theapproach makes it possible to evaluate the efficacy and toxicity of theTwHF extract and control the quality and safety of the preparation ofthis material for clinical trials and animal experiments.

EXAMPLE 16 TwHF Extract Interferes With Progesterone Metabolism

The present prophetic example describes a property of the TwHF extractin interfering with progesterone metabolism. It is expected that theextract or components thereof may bind the progesterone receptor as manycompounds that bind to the glucocorticoid receptor also bind to theprogesterone receptor and therefore be used as a means of birth controlor as a means of interrupting a pregnancy. As shown in FIG. 29D, RU486inhibits endotoxin-induced PGE₂ production by human peripheral bloodmonocytes, however, it is not as effective as the TwHF extract at lowerconcentrations. The use of TwHF preparations in humans is provided inExample 9.

EXAMPLE 17 Therapeutic Preparation Of The TwHF Extract

The present example details convenient preparations of the extracts,such as pills, tablets, capsules, and the like that may be prepared andused in the various aspects of the invention.

The TwHF extract, or purified components thereof, may be orallyadministered, for example, with an inert diluent or with an assimilableedible carrier, or they may be enclosed in hard or soft shell gelatincapsule, or they may be compressed into tablets, or they may beincorporated directly with the food of the diet. For oral therapeuticadministration, the extract, or components thereof, may be incorporatedwith excipients and used in the form of ingestible tablets, buccaltables, troches, capsules, elixirs, suspensions, syrups, wafers, and thelike. Such compositions and preparations should contain at least 0.1% ofactive compound. The percentage of the compositions and preparationsmay, of course, be varied and may conveniently be between about 2 toabout 60% of the weight of the unit. The amount of active compounds insuch therapeutically useful compositions is about 30-120 mg, preferablyabout 60 mg, such that a suitable dosage will be obtained.

The tablets, troches, pills, capsules and the like may also contain thefollowing: a binder, as gum tragacanth, acacia, cornstarch, or gelatin;excipients, such as dicalcium phosphate; a disintegrating agent, such ascorn starch, potato starch, alginic acid and the like; a lubricant, suchas magnesium stearate; and a sweetening agent, such as sucrose, lactoseor saccharin may be added or a flavoring agent, such as peppermint, oilof wintergreen, or cherry flavoring. When the dosage unit form is acapsule, it may contain, in addition to materials of the above type, aliquid carrier. Various other materials may be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules may be coated with shellac, sugar or both. Asyrup of elixir may contain the active compounds sucrose as a sweeteningagent methyl and propylparabens as preservatives, a dye and flavoring,such as cherry or orange flavor. Of course, any material used inpreparing any dosage unit form should be pharmaceutically pure andsubstantially non-toxic in the amounts employed. In addition, theextract, or components thereof, may be incorporated intosustained-release preparation and formulations.

The extract, or components thereof, related to triptolide, tripdiolide,or wilforonide selected by the biological and molecular activitiesdescribed herein may also be administered parenterally orintraperitoneally. Solutions of the purified active compounds as freebase or pharmacologically acceptable salts can be prepared in watersuitably mixed with a surfactant, such as hydroxypropylcellulose.Dispersions can also be prepared in glycerol, liquid polyethyleneglycols, and mixtures thereof and in oils. Under ordinary conditions ofstorage and use, these preparations contain a preservative to preventthe growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), suitable mixtures thereof,and vegetable oils. The proper fluidity can be maintained, for example,by the use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the extract,or components thereof, in the required amount in the appropriate solventwith various of the other ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the various sterilized active ingredients into asterile vehicle which contains the basic dispersion medium and therequired other ingredients from those enumerated above. In the case ofsterile powders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

EXAMPLE 18 Methods For Screening For Agents Having GR Binding ActivityAnd A Steroid-Sparing Effect

The present example provides assays for screening candidate substancesfor glucocorticoid receptor binding activity while being inactive forinduction of steroid responsive genes. Since TwHF, and componentsthereof, are demonstrated for the first time in the present disclosureto bind the receptor even in the presence of dexamethasone; an assay foridentifying other glucocorticoid receptor binding substances has beenidentified using TwHF, or a receptor-active fraction/component thereof.By way of example, a candidate substance is incubated with theglucocorticoid receptor in the presence of TwHF, or a receptor-bindingcomponent thereof, and substances competing with TwHF, or a componentthereof, for the receptor would be selected. It is contemplated thatthis screening technique will prove useful in the general identificationof a compound or mixture of compounds that has binding activity for theglucocorticoid receptor. In other embodiments of the method, thecandidate substance will be further screened to determine if it iscapable of inhibiting the activation of steroid dependent genes, usingthe TwHF as a standard.

Another embodiment of the present invention is a method for determiningthe ability of a candidate substance to bind the glucocorticoid receptorin, for example, a competitive binding assay in the presence of TwHFpreparation, or pharmacologically active glucocorticoid receptor bindingcomponents thereof. For example, the method, in some embodiments,includes generally the steps of obtaining a glucocorticoid receptorpreparation, admixing a candidate substance with the glucocorticoidreceptor preparation in the presence of TwHF preparation or apharmacologically active component thereof that binds glucocorticoidreceptor, and determining the ability of the candidate substance to bindthe glucocorticoid receptor in the presence of the TwHF, orpharmacologically active component thereof.

Naturally, one would measure or determine the binding of the TwHFcomposition or component of the composition, in the absence of the addedcandidate substance in a separate control study. One would then add thecandidate substance and the TwHF preparation together to a receptorpreparation and determine the ability of the candidate substance tocompete with the TwHF for binding the receptor. A candidate substancewhich reduces the binding of TwHF preparation to the receptor relativeto the binding in its absence is indicative of a candidate substancewith glucocorticoid receptor binding capability.

Accordingly, in screening assays to identify pharmaceutical agents thatbind the glucocorticoid receptor, it is proposed that compounds isolatedfrom natural sources such as plants, animals or even sources such asmarine, forest or soil samples, may be assayed for the presence ofpotentially useful pharmaceutical agents. It will be understood that thepharmaceutical agents to be screened could also be derived from chemicalcompositions or man-made compounds. The active compounds may includefragments or parts of naturally-occurring compounds or may be only foundas active combinations of known compounds which are otherwise inactive.

Any method may generally be employed to determine glucocorticoidreceptor binding. For example, by using the methods of Example 15 todetermine the amount of unbound triptolide or tripdiolide present in asupernatant fraction of the above assay, it would be possible todetermine the amount of binding by the candidate substance. A controlsample would include a mixture where only TwHF was present.

Further methods will be those in which the glucocorticoid receptorincorporates or is conjugated to a label, such as an enzymatic, chemicalor radiolabel, or incorporates one of the ligands of a two ligand-baseddetection system such as the avidin/biotin system. For ease and safety,the use of enzymatic labels, such as, for example, horseradishperoxidase, urease or alkaline phosphatase is preferred. In such cases,a colorimetric indicator substrate would be employed to provide a meansdetectable by the human eye or by a spectrophotometer.

One of skill in this art upon reading the present disclosure willrealize that a candidate substance that is able to bind theglucocorticoid receptor in the presence of TwHF or at least onecomponent thereof possessing pharmacological activity for bindingglucocorticoid receptor, may activate steroid responsive genes or mayinhibit the activation of steroid responsive genes. An assay using areporter gene under the control of glucocorticoid receptor regulatoryregions as used in Example 13 would determine whether steroid responsivegenes are activated, and also serve to provide a method for furtherselection among candidate substances for those which do not activatesteroid responsive genes.

An example of a glucocorticoid receptor preparation is a human skinfibroblast preparation. An example of GR regulatory region is the GRinducible elements of the MMTV long terminal repeat. An example of areporter gene is the luciferase gene, or the CAT gene (chloramphenicolacetyltransferase).

As used herein, "pharmaceutically acceptable carrier" includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like. The use ofsuch media and agents for pharmaceutical active substances is well knownin the art. Except insofar as any conventional media or agent isincompatible with the active ingredient, its use in the therapeuticcompositions is contemplated. Supplementary active ingredients can alsobe incorporated into the compositions.

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What is claimed is:
 1. A method of selecting a substance for treating inflammation or immune disease comprising:admixing a candidate substance with a glucocorticoid receptor preparation which is capable of binding the candidate substance in the presence of a Tripterygium wilfordii Hook F extract or a glucocorticoid receptor binding component thereof; determining if the candidate substance binds to the glucocorticoid receptor; selecting the candidate substance which binds to the glucocorticoid receptor; determining if the bound candidate substance induces glucocorticoid responsive gene expression; and selecting the candidate substance which binds to the glucocorticoid receptor but which does not induce glucocorticoid responsive gene expression as the substance for treating inflammation or immune disease.
 2. The method of claim 1 wherein the glucocorticoid receptor binding component is triptolide, tripdiolide or wilforonide.
 3. The method of claim 1 where the determining if the bound candidate substance induces glucocorticoid responsive gene expression is carried out using a reporter gene construct comprising a reporter gene under regulatory control of a glucocorticoid responsive element.
 4. The method of claim 3 where the glucocorticoid responsive element is from the MMTV long terminal repeat.
 5. The method of claim 3 where the reporter gene is the luciferase gene or the chloramphenicol acetyltransferase gene.
 6. The method of claim 1 wherein the glucocorticoid receptor is from a human skin fibroblast preparation. 